Detecting complement activation

ABSTRACT

Methods of detecting complement activation including steps of detecting in a sample from a subject a level of iC3b wherein the detecting involves specific interaction between the iC3b and a non-cross-reactive antibody thereto, comparing the detected level with a reference level, which reference level is within a range of about 10 ng/ml to about 5,000 ng/ml, wherein determination that the detected level is above the reference level indicates that the subject is suffering from or susceptible to undesirable and/or pathologic complement activation, and administering treatment to treat undesired complement activation if the detected level is above the reference level. Other methods of detecting complement activation with or without measuring iC3b are also provided.

RELATED APPLICATIONS

This application is a divisional of U.S. Non-Provisional applicationSer. No. 13/461,709, filed May 1, 2012, which is a continuation-in-partof U.S. Non-Provisional application Ser. No. 13/287,432, filed Nov. 2,2011, which claim priority under 35 U.S.C. 119§(e) to United States toU.S. Provisional Application Ser. No. 61/409,297, filed Nov. 2, 2010,the contents of which are incorporated by reference herein in theirentirety.

BACKGROUND

Inflammation is the physiological response of vascularized tissue toinjury, infection, and certain diseases. The inflammatory process is abiological requirement for wound healing after traumatic injury and forthe clearance of infection. However, inflammation can also damageself-tissue. For this reason, inflammation is often considered adouble-edged sword.

Complement is the most ancient arm of the immune system and is deeplyrooted in the inflammatory process. The complement protein cascade is afirst line of defense against invading microbes and a critical player inthe wound healing process. The complement cascade involves more than 30serum and cellular proteins and plays important roles in innate andadaptive immunity. Complement activation can occur via three majorpathways: the classical, alternative, and lectin pathways. All threemajor pathways of complement activation converge on the central proteincomplement component 3 (C3). C3 is a central mediator of inflammationand is activated by most factors that cause inflammation. FIGS. 1 and 2provide schematic overviews of C3 and its activation products.

Complement, and C3 in particular, is associated with several diseaseindications, both acute and chronic. Examples include, but are notlimited to, trauma, respiratory distress, sepsis, other forms ofinfection, infectious diseases (e.g., hemorrhagic fevers), multipleorgan failure, age-related macular degeneration, rheumatoid arthritis,systemic lupus erythematosus, glomerular nephritis, ischemia,reperfusion injury, inflammatory bowel disease, intracranial hemorrhage,myocardial infarction, and cardiac arrest.

Several reports have shown that complement activation occurs immediatelyafter injury and correlates with severity of injury. In one study,circulating levels of complement protein in trauma patients were foundto correlate with patient outcome. See Hecke, et al., Circulatingcomplement proteins in multiple trauma patients—correlation with injuryseverity, development of sepsis, and outcome, Crit. Care Med. 25(12):2015-24 (1997). In this study, the authors measured the plasmaconcentrations of both C3a and total C3 directly after the injury and inthe ICU in the days following injury. They detected evidence ofcomplement C3 activation at the earliest time points following injury.However, complement activation was more pronounced in non-survivors thansurvivors for the first eight hours. At the earliest time points, thedegree of C3 activation correlated with patient outcome. Hecke et al.also found the ratio of the C3 split product, C3a, when taken as a ratioto total complement, was a better predictor of outcome than C3a alone.

A similar study by Zilow, et al., Complement activation and theprognostic value of C3a in patients at risk of adult respiratorydistress syndrome, Clin. Exp. Immunol. 79: 151-57 (1990),retrospectively found that monitoring of C3a and total C3 at frequent (6hr) intervals might be useful for identifying patients at high risk foror in the early stages of respiratory distress. These investigators drewthe first plasma sample within 2 hours of injury and repeated 6 hoursamplings for the first 48 hours and then at daily intervals thereafter.Zilow et al. found a significant correlation between C3a levels andC3a:total C3 ratio at 6 and 12 hours, as well as from 5 days outward.

In the field of trauma care, the first hour after injury is sometimesreferred to as the “Golden Hour.” While not desiring to be bound bytheory, it is generally believed that intervention within the first hourafter traumatic injury greatly increases the outcome of the patient.Better diagnostic information provided earlier would help improve thecritical care specialist's intuition when making treatment decisions.

SUMMARY OF THE INVENTION

The present inventors have developed new methods and assays for thequalitative and quantitative measurement of native or intact C3 and/oriC3b. The present inventors further provide point-of-careimplementations of such methods and assays.

Provided methods and assays are suitable for use, for example, inmedicine, including in diagnostics and therapeutics, and particularly inclinical settings. Among other things, provided methods and assays aresuitable for use in directing patient care at the earliest time pointsimmediately following traumatic injury or other physiological crises.Alternatively or additionally, provided methods and assays are suitablefor use, for example to select, monitor, and/or adjust treatments forpatients suffering from or susceptible to one or more diseases statesaffected by presence or level of complement activation. Such diseasestates include, for example, autoimmune diseases such as systemic lupuserythematosus.

By carefully selecting, capturing and detecting antibody pairs thatavoid interfering cross-talk, the inventors have surprisingly found thatit is possible to detect and quantify biomarkers of complementactivation including native or intact C3 and/or iC3b, while avoiding thefalse positive results that have plagued more conventional testingmethods for these analytes. This invention may be applied in a varietyof formats; the inventors have further found that a lateral flow assaytype format, and particularly a lateral flow immunoassay format,provides particular advantages and surprising features.

While the complement response is an important defense system againstdisease, several features make accurate measurement of complementactivation difficult, particularly in a clinically relevant time period.Known technologies require an hour, and often two hours or more todetermine complement levels. During this time, significant deteriorationof a patient's condition may be occurring that does not visibly manifestuntil significant, or even irreversible, damage has occurred.Additionally, complement proteins are known to be easily activated byhandling and other experimental conditions. The Applicants havediscovered, among other things, that levels or types of handling notpreviously appreciated to activate complement indeed can significantlyaffect assay results. Perhaps most surprisingly, Applicants demonstrateherein that the passage of time is a significant factor in spontaneouscomplement activation, even without handling.

The present invention provides technologies that address thesepreviously unidentified sources of problems. For example, in someembodiments, the present invention provides methods in which relevantsteps are all performed within a restricted time period. According tothe present invention, such methods provide advantages includingminimizing spontaneous complement activation and, alternatively oradditionally, providing clinically relevant data within a time period,measured from initiation of sample collection from a subject, that ismaterially reduced as compared with standard methodologies. In someembodiments, provided assays are completed within a time period,measured from initiation of sample collection from a subject, that isless than about 60 minutes or fewer, about 50 minutes or fewer, about 40minutes or fewer, about 30 minutes or fewer, about 20 minutes or fewer,about 10 minutes or fewer, or about 5 minutes or fewer. By contrast,many prior art assays do not provide data for hours. Such a differencein time can make a life or death difference to a patient.

The present invention further identifies a previously unappreciatedsource of a problem with prior art assays that detect changes (e.g.,decreases) in total complement protein levels. The rationale behind suchprior art assays is that injury or disease can trigger massivecomplement activation, resulting in decrease in total complement proteinlevels. However, the present invention encompasses the finding thathigher levels of complement activation than previously appreciated canbe triggered within an individual by non-pathogenic events such asexercise, and also within a sample by handling, or even by the passageof time. The present invention therefore reveals that prior art assayslack specificity in that they cannot distinguish the reason thatcomplement proteins are reduced, or the fate of relevant complementproteins. By contrast, embodiments of the present invention provideassays that permit discrimination between undesirable and/orpathological and non-undesirable and/or pathological complementactivation. For example, some such provided assays measure levels ofintact C3 and/or of one or more activation products, such as iC3b.Assessment of complement protein levels, rather than total complementprotein, in accordance with the present invention, distinguishesundesirable and/or pathologic complement activation from a generaldecrease in circulating complement protein, for example as may occur inresponse to lifestyle changes or the like.

In one aspect, the present invention provides methods including steps ofdetecting in a sample from a subject a level of intact C3, wherein thedetecting involves specific interaction between the intact C3 and anon-cross-reactive antibody thereto, comparing the detected level with areference level, which reference level is within a range of about 350ug/ml to about 1,700 ug/ml, wherein determination that the detectedlevel is below the reference level indicates that the subject issuffering from or susceptible to undesirable and/or pathologiccomplement activation, and optionally administering treatment if thedetected level is below the reference level.

In one aspect, the present invention provides methods including steps ofdetecting in a sample from a subject a level of iC3b, wherein thedetecting involves specific interaction between the iC3b and anon-cross-reactive antibody thereto, comparing the detected level with areference level, which reference level is within a range of about 10ng/ml to about 5,000 ng/ml, wherein determination that the detectedlevel is above the reference level indicates that the subject issuffering from or susceptible to undesirable and/or pathologiccomplement activation, and optionally administering treatment if thedetected level is above the reference level.

In one aspect, the present invention provides methods steps of detectingin a sample a ratio of intact C3 level to iC3b level, wherein thedetecting involves specific interaction between the intact C3, the iC3b,or both with a non-cross-reactive antibody thereto, comparing thedetected level with a reference ratio of about 0.001 whereindetermination that the detected level is below the reference levelindicates that the subject is suffering from or susceptible toundesirable and/or pathologic complement activation, and optionallyadministering treatment if the detected level is below the referencelevel.

These and other objects, features, embodiments, and advantages willbecome apparent to those of ordinary skill in the art from a reading ofthe following detailed description and the appended claims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 provides a schematic overview of the complement system.Complement is activated by three major pathways, all of which convergeon the activation of intact C3. Proteolytic activation of C3 producesthe C3 split products C3a and C3b. C3b is further proteolyticallymodified to form iC3b, a biomarker for C3 activation. Intact C3 and iC3bare circled in the schematic.

FIG. 2 provides a schematic representation of C3 activation anddeactivation. Intact C3 is activated proteolytically to C3a and C3b.Some C3b molecules covalently attach to surfaces; others react withwater and stay in circulation. C3b is deactivated by the proteaseFactor 1. The first deactivation product is iC3b, which is formed by theactivity of Factor 1 removing a short peptide, C3f. iC3b is furtherdegraded to C3c and C3dg, the latter ultimately being degraded to C3d.

FIG. 3 is a schematic representation of specific recognition of intactC3 and iC3b by antibody pairs. (A) Intact C3 is recognized by twoantibodies: a first antibody recognizes C3a, which is only present inthe intact C3 molecule; a second antibody recognizes a region in C3dthat is present in both intact C3 and iC3b. The second antibodyparticipates in distinguishing C3 and its derivatives from other proteinmolecules but not intact C3 from iC3b. An alternate pair of antibodiesfor intact C3 include antibodies that recognized C3a and C3f. The upperpanel shows that in intact C3, while epitopes “A”, “F” and “D” can bedetected, the “Y” and “G” epitopes buried, and thus not detected. Thelower panels shows that after activation by C3 convertase, C3a isremoved and C3b, being short lived (2 min.) is not a target. (B) TheiC3b protein is recognized by another antibody pair. The first antibodycontacts the protein at a neoepitope believed to be located near the C3gregion. This epitope is revealed once Factor I removes the C3f fragment.The neoepitope is occluded once Factor I degrades iC3b to C3c and C3dg.The second antibody recognizes the C3d epitope (i.e., recognizingepitopes D and G). An alternate pair for iC3b includes theaforementioned iC3b antibody and a second that recognizes activated C3d(i.e., recognizing epitopes Y and G) (Quidel® A250).

FIG. 4 is a schematic of one embodiment of a lateral flow immunoassay ofthe present invention.

FIG. 5 is a schematic representation of two embodiments of a lateralflow immunoassay in accordance with the present invention. (A) shows alateral flow immunoassay for detection of a single analyte. (B) shows alateral flow immunoassay for detection of two separate analytes (intactC3 and iC3b) in parallel membrane strips.

FIG. 6 is a depiction of three embodiments of single analyte lateralflow immunoassays. (A) shows a test cassette for a total C3 lateral flowimmunoassay. (B) shows a test cassette for an intact C3 lateral flowimmunoassay. (C) shows a test cassette for an iC3b lateral flowimmunoassay.

FIG. 7 is a depiction of two embodiments of a double analyte lateralflow immunoassay test cassette, for the assessment of intact C3 andiC3b. (A) shows a test cassette comprising two separate ports for sampleloading and two membrane strips in parallel, one for each analyte. (B)shows a test cassette with a single port for sample loading and twomembrane strips in parallel, one for each analyte.

FIG. 8 is a depiction of two embodiments of a triple analyte lateralflow immunoassay test cassette, for the assessment of total C3, intactC3, and iC3b. (A) shows a test cassette comprising three separate portsfor sample loading and three membrane strips in parallel, one for eachanalyte. (B) shows a test cassette with a single port for sample loadingand three membrane strips in parallel, one for each analyte.

FIG. 9 is a schematic representation of two embodiments of a lateralflow immunoassay. (A) shows a lateral flow immunoassay for detection ofa single analyte. (B) shows a lateral flow immunoassay for detection oftwo separate analytes (intact C3 and iC3b) in series on the samemembrane strip.

FIG. 10 is a depiction of two embodiments of a lateral flow immunoassayfor multiple analytes in series. (A) shows a lateral flow immunoassayfor detection of two analytes and a control in series on the samemembrane strip. (B) shows a lateral flow immunoassay for detection ofthree analytes and a control in series on the same membrane strip.

FIG. 11 shows a comparison of sensitivities, dynamic range, test-to-testvariability, and assay time for three embodiments of the instant lateralflow immunoassays. (A) shows a standard curve graph for a test stripdetecting iC3b without a cassette casing. (B) shows a standard curvegraph of an embodiment of the lateral flow immunoassay wherein the teststrips are enclosed in a cassette, which allows more controlledadministration of the test sample volume. Concentration of antibodysolution used for gold conjugation is 0.5 mg/ml and BSA is included inthe reaction mixture. (C) shows a standard curve graph of anotherembodiment of a test strip integrated into a cassette, wherein theconcentration of antibody solution used for gold conjugation is 1 mg/mland BSA is removed from the reaction mixture. Sensitivity of the assayreaches 10 ng/ml with a dynamic range extending to 10 ug/ml.

FIG. 12 shows sensitivity of an embodiment of a lateral flow immunoassayfor iC3b. Sensitivity ranges from 10 ng/ml to 10 ug/ml. Standard erroris less than 3% at 20 minutes. R square=0.9892.

FIG. 13 shows sensitivity of an embodiment of a lateral flow immunoassayfor intact C3. Sensitivity ranges from 20 ng/ml to 10 ug/ml. Standarderror is less than 3% at 20 minutes. R square=0.9964. Error bars areshown, but are smaller than the plotted points.

FIG. 14 shows crosstalk between intact C3 and iC3b antibodies inexemplary lateral flow immunoassays.

FIG. 15 shows intact C3 and iC3b levels in basal tears from a singleindividual at 12 hour intervals, as assayed by exemplary lateral flowimmunoassays described herein.

FIG. 16 shows intact C3 and iC3b levels in whole blood from a normalhealthy individual. Results show approximately 2500-fold more intact C3than iC3b in whole blood from a healthy donor.

FIG. 17 shows intact C3 and iC3b levels in whole blood from a healthyindividual. Results show approximately 333-fold more intact C3 than iC3bin whole blood from a healthy donor.

FIG. 18 shows intact C3 and iC3b levels in a normal individual 2 hoursafter heavy exertion (100 mile bicycle ride). Results show greater than1000-fold more intact C3 than iC3b in whole blood from a healthyindividual post-exertion.

FIG. 19 is a table of exemplary body fluids suitable for use with theassays and methods disclosed herein.

FIG. 20 shows two schematic representations of lateral flow embodimentsof the present invention that may be used to measure a single analytefrom a sample. (A) shows a schematic of an exemplary embodiment of thepresent invention, and (B) shows a schematic of an exemplary processaccording to aspects of the present invention.

FIG. 21 depicts an exemplary method of carrying out one or more aspectsof the present invention. Also shown are exemplary illustrations of howthe method may look when carried out, as well as pictures of assay teststrips and a test strip reader in accordance with aspects of the presentinvention. An exemplary protocol is described below: 1. Clean fingerusing an alcohol swab; 2. Prick finger with lancet; 3. Squeeze fingergently, and collect by capillary action 10 ul of blood using theMICROSAFE® Tube; 4. Expel the blood sample directly into CompAct Test Asample port of cassette; 5. Immediately expel 3 drops of assay bufferinto sample port to mix with blood; 6. Set time for 15 or 20 minutes; 7.Repeat assay from same finger stick of blood using CompAct Test Bcassette; 8. Before the 15 or 20 minutes is up, turn the Kypha CompActReader on 9. Assess each test before reading to make sure that theControl line is readily visible and that smearing is not an issue; 10.When the timer is up, slide down cover and place CompAct Test A intoreader; 11. Slide cover back up and hit start button. Reader willdisplay results after about 15 seconds; 12. Capture results. Reader canstore up to 250 tests; 13. Discard CompAct Test A and replace withCompAct Test B; 14. Hit start button; and 15. Capture results anddiscard CompAct Test B.

FIG. 22 shows photographs of assay test strips that were used inaccordance with certain embodiments of the invention, as described inExamples, to generate the standard curves in FIG. 23. Each data point inFIG. 23 was generated from triplicate measurements shown in this FIG.22.

FIG. 23 shows a several standard curves generated according to methodsof the present invention showing levels of native C3 protein at 10minutes, 20 minutes, and 30 minutes following sample preparation.

FIG. 24 shows variance in measured values for native C3 for the samplesshown in FIG. 22 and the data shown in FIG. 23.

FIG. 25 shows photographs of the assay test strips that were used togenerate the standard curves in FIG. 26. Each data point in FIG. 26 wasgenerated from triplicate measurements shown in this figure.

FIG. 26 shows a several standard curves generated according to methodsof the present invention showing levels of iC3b protein at 10 minutes,20 minutes, and 30 minutes following sample preparation.

FIG. 27 shows triplicate measures of (A) native C3 and (B) iC3b from asingle blood sample as assayed according to methods of the presentinvention.

FIG. 28 shows measurements of native C3 levels from three donors asmeasured in whole blood, serum, and plasma according to methods of thepresent invention.

FIG. 29 shows measurements of iC3b levels in plasma or serum as measuredby methods of the present invention and compared to known ELISA methods.Panels (A), (B), and (C) show data by donor in bar graph form, while (D)shows the data from all three donors in tabular form.

FIG. 30 shows iC3b levels from samples of purified iC3b protein asmeasured by methods of the present invention and compared to known ELISAmethods, and shows a comparison to data generated from a sample ofplasma using either an assay of the present invention or a traditionalELISA.

FIG. 31 shows levels of iC3b protein as measured from samples of wholeblood, plasma, or serum from two donors. The assays were run either whenthe samples were fresh or when the samples had been exposed to roomtemperature for four hours. The Y-axis is in log scale, thusdemonstrating that there is approximately 100-fold increase in iC3blevels after samples are incubated room temperature for >4 hr prior totesting. These results indicate that substantial increases in iC3blevels occur rapidly at RT, raising the possibility that substantial C3activation may be occurring in standard iC3b ELISA assays. Samplesstored at −80° C. do not differ substantially from fresh samples (notshown).

FIG. 32 shows the effect of time on measured iC3b levels on samples ofwhole blood (labeled “Blood”) as compared to an iC3b standard curve(labeled “iC3b”). Samples were exposed to room temperature for (A) 5minutes, (B) 10 minutes, (C) 15 minutes or (D) 20 minutes.

FIG. 33 shows measured iC3b levels in samples of plasma, or serum fromthree donors. Samples from each donor were exposed to one of twoconditions, either exposure to room temperature for five (5) minutes orexposure to room temperature for sixty (60) minutes. Levels of iC3b weremeasured at six intervals during the exposure time and the levels werenormalized to an iC3b standard curve for direct comparison purposes.

FIG. 34 shows the effect of time on the generation of iC3b in humanplasma samples. (A) shows a photo of lateral flow test strips inaccordance with aspects of the invention, and (B) shows a graph of theiC3b generation over time as read from the strips shown in (A).

FIG. 35 depicts iC3b activation in human plasma and serum samples after(A) 2 minutes of incubation and (B) 5 minutes of incubation using anELISA format assay.

FIG. 36 shows a bar chart of iC3b activation in samples of human serumafter 2 minutes, 5 minutes, 15 minutes, or 60 minutes of incubation inan ELISA format assay.

FIG. 37 shows human serum-derived C3 deposition and activation overtime, in the absence of an antigen:antibody complex.

FIG. 38 shows human serum-derived C3 deposition and activation overtime, in the absence of antibody-coated wells.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Heretofore, a point-of-care assay for measuring complement activationwithin the actionable window of treatment has not been known in the art.Although associations between complement and disease or trauma have longbeen recognized, C3 is monitored in only a small number of diseases orconditions. Even in those instances, current assay methods havelimitations. First, in most cases, traditional complement assays aredirected to total C3 as the target analyte (for example, via turbidityassays and ELISA). Total C3 is a combination of intact (or native) C3and C3 activation and deactivation products. These tests generallydetect decreases in circulating total C3 levels. Decreased levels oftotal C3 therefore only measure C3 depletion due to massive activation.However, other factors such as diet or exercise can cause lower steadystate levels of C3. As total C3 assays do not measure turnover, thecauses of activation cannot be distinguished. Furthermore, a test thatmeasures total C3 cannot monitor the real-time changes in C3 activationsignature that would be useful in directing patient care. For example,patients suffering from trauma or systemic lupus (marked by decreased C3levels) would benefit from improved C3 activation monitoring. Currently,treatment effectiveness for systemic lupus is measured by a return ofdepressed C3 levels to normal levels. However, the physician hasdifficulty in discerning whether the underlying disease process has beenhalted or just retarded sufficiently for homeostatic mechanisms toreturn C3 to physiologically normal levels.

A second limitation in current C3 testing is the time required toperform most assays. A typical ELISA assay for the detection ofcomplement activation requires hours to perform and the readyavailability of a laboratory and a skilled technician. This assayplatform is therefore not useful for indications of inflammatorydysfunction, in which biomarkers change on the order of minutes andclinical intervention is required on a similar timescale.

A third limitation in current C3 testing lies in the nature of theprotein cascade itself. Complement is notoriously fastidious and canbecome activated by virtue of standard analysis procedures (handling,storage, and exposure to foreign materials that contact C3 duringanalysis). Complement is very effective at lysing invading microbes andinitiating the wound healing response at sites of injury. Thiseffectiveness is due in part to the ability of C3 to be activated byforeign materials such as bacterial cell wall components. While thisproperty is useful in directing an immune response to new foreignpathogens, this same property presents formidable challenges toexperimental and diagnostic study. Materials such as plastics used insample handling, manipulation of the sample itself, and improper storageconditions can also trigger complement activation. The more processingand handling steps required to perform a given assay, the more falsepositives can be expected, due to activation of complement by virtue ofthe assay itself. Such false positives complicate traditional testingand render current testing methods unsuitable for use in directingpatient care in near real-time.

A further consideration in complement activation testing is theselection of the best biomarker for detecting real time changes in theinflammatory response. C3 has several attractive qualities as abiomarker in inflammation. First, as the central protein of thecomplement system, C3 is activated by most stimuli that will causecomplement activation. Second, C3 activates in proportion to the degreeof injury or infection. Third, C3 responds in near real-time to aphysiological insult. Complement activation occurs in direct response toan agent causing crisis, in contrast to other acute phase inflammatorymarkers that take hours or days to respond. This rapid response propertyis not present in other biomarkers frequently used in the clinic.

Specifically, intact (or native) C3 is a valuable marker of inflammatorystatus. Intact C3 represents the amount of C3 available for activation.Total C3 represents intact C3 as well as all C3 activation products. Atpresent, standard complement assays generally measure total C3 viaturbidity assays or ELISA. Although technically easier to perform, totalC3 assays cannot detect C3 depletion as accurately as intact C3 assays.Monitoring intact C3, especially over time, is useful for followingmassive complement activation events, such as those that occur in traumaand other systemic complement activation indications. Monitoring intactC3 over time allows a clinician to detect the onset of animmunosuppressive state caused by depletion of C3. Further, intact C3may be more useful than total C3 when calculating complement activationindexes. Intact C3 assays have historically proven difficult toadminister or depend upon, in part because intact C3 is very labile andcan denature or self-activate if not handled properly.

The C3 split product, iC3b, is also a valuable marker of inflammatoryresponse. iC3b has a half-life of 30 to 90 minutes, serving as a lessvolatile (compared to C3a), but still rapidly responsive biomarker.However, iC3b is present at much lower levels than intact C3 in patientsamples. Even a small degree of cross-talk (for example 1%) betweenintact C3 protein and the iC3b-specific assay produces a false positiveiC3b signal at a level twice that of normal circulating iC3b. Hence,while a desirable marker of inflammation, heretofore iC3b has posedsignificant challenges in diagnostic testing.

WO 2010/135717, by Zhang et al., published Nov. 25, 2010, is directed tomethods for assessing complement activation via the biomarkers intactC3, iC3b, and total C3. However, Zhang et al. is limited to traditionalsandwich-type immunoassays such as ELISA, requiring laboratoryprocessing and the expertise of skilled technicians. Further, the assaysand methods of Zhang et al. require sample preparation, storage, andhandling steps that are known to activate the labile intact C3 producefalse positive test results, impeding the ability to accurately measureintact C3. Moreover, the assays and methods of Zhang et al. requirehours to process and are thus incapable of providing the near real-timedata that can impact patient care in the earliest time points afterphysiological crisis.

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document.

While the following terms are believed to be well understood by one ofordinary skill in the art, definitions are set forth to facilitateexplanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently disclosed subject matter belongs.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

“Analyte” means any entity, particularly a chemical, biochemical orbiological entity to be assessed, e.g., whose amount (e.g.,concentration or mass), activity, composition, or other property(ies)is/are to be detected, measured, quantified, evaluated, analyzed, etc.An “analyte” can be a single molecular species or can be composed ofmultiple distinct molecular species.

“Antibody” encompasses intact and/or full length immunoglobulins oftypes IgA, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgE, IgD, IgM, IgY,antigen-binding fragments or single chains of complete immunoglobulins(e.g., single chain antibodies, Fab fragments, F(ab′)2 fragments, Fdfragments, scFv (single-chain variable), and dAb fragments), and otherproteins that include at least one antigen-binding immunoglobulinvariable region, e.g., a protein that comprises an immunoglobulinvariable region, e.g., a heavy (H) chain variable region (VH) and alight (L) chain variable region (VL). The light chains of an antibodymay be of type kappa or lambda. An antibody may be polyclonal ormonoclonal. A polyclonal antibody contains immunoglobulin molecules thatdiffer in sequence of their complementarity determining regions (CDRs)and, therefore, typically recognize different epitopes of an antigen.Often a polyclonal antibody is derived from multiple different B celllines each producing an antibody with a different specificity. Apolyclonal antibody may be composed largely of several subpopulations ofantibodies, each of which is derived from an individual B cell line. Amonoclonal antibody is composed of individual immunoglobulin moleculesthat comprise CDRs with the same sequence, and, therefore, recognize thesame epitope (i.e., the antibody is monospecific). Often a monoclonalantibody is derived from a single B cell line or hybridoma. An antibodymay be a “humanized” antibody in which for example, a variable domain ofrodent origin is fused to a constant domain of human origin or in whichsome or all of the complementarity-determining region amino acids oftenalong with one or more framework amino acids are “grafted” from arodent, e.g., murine, antibody to a human antibody, thus retaining thespecificity of the rodent antibody.

In another embodiment, the capturing and/or detecting agents includeother ligands, such as natural receptors for activated C3 (e.g.,complement receptors 1, 2, and 3), aptamers, peptides, other smallmolecule ligands, and the like.

An aspect of certain embodiments of the invention is the selection ofantibodies for use as capture and detection agents. The inventorsdiscovered that many published and commercially available antibodiesexhibited crosstalk between intact C3 and various C3 cleavage productsor between different C3 cleavage products. For example, certainmonoclonal antibodies against human C3a show significant and unexpectedcross-reactivity with C3b and iC3b. It was recognized thatcross-reactivity could be a significant source of inaccuracy in certainsituations. Of particular concern in developing an assay for iC3b wascrosstalk between intact C3 and iC3b observed with many of the iC3bantibodies tested. Further testing showed that crosstalk between C3b andiC3b was even more significant with at least some of these antibodies.This was of concern because iC3b levels are expected to be present atmuch lower levels than intact C3 in patient samples. One aspect ofcertain embodiments of the invention is the selection of antibodies withspecificity for intact C3 or iC3b so as to minimize such crosstalk. Incertain embodiments, antibodies with specificity for intact C3 or iC3bare not substantially cross-reactive. In this context, “notsubstantially cross-reactive” means less than about 0.1% cross-reactive,meaning that a 1 ug/ml solution of C3 must register as less than about 1ng/ml of iC3b. The about 0.1% threshold is based on the physiologicallevels of intact C3 and iC3b in a normal individual. Normal iC3b levelsare approximately 0.5% that of total C3 in circulation. If C3 crosstalkcontributes more than about 25% to the iC3b signal in a complementactivation assay, the assay can produce false positive results thatabrogate the utility of the assay.

“Body fluid” means any fluid in the body that may be assayed forcomplement activation. Body fluids include, but are not limited to,whole blood, serum, plasma, urine, tears, saliva, wound exudate,broncheoalveolar lavage fluid, and cerebrospinal fluid. See FIG. 19 fora non-limiting list of suitable body fluids.

“Complement activation level” means the amount of complement (generallyC3) that is activated at a given time point. Amounts (i.e., levels) ofintact C3, iC3b, and/or total C3 are typically expressed in terms ofconcentration but may be expressed in terms of mass or weight.Concentration may be expressed in various ways, e.g., in terms ofmolarity, molality, mole fraction, mass fraction (mass of a substance ina mixture as a fraction of the mass of the entire mixture), mass perunit volume, etc. For purposes of description herein, concentration(e.g., mass per unit volume) will generally be used. Complementactivation level can also be described as a ratio of iC3b to intact ortotal C3, or as a ratio of C3a to total C3.

“Complement-associated disorder,” as used herein, refers to a disorderor condition characterized by a modification in complement activation.Examples of complement associated disorders include, but are not limitedto, trauma, such as traumatic brain injury, spinal cord injury, surgery,and intracranial pressure; inflammatory distress, such as severeallergies, systemic inflammatory response syndrome (SIRS), multipleorgan failure (MOF), acute or adult respiratory distress syndrome(ARDS), septic shock, and shock; paroxysmal nocturnal hemoglobinuria(PNH); hereditary angiodema; renal disease, such as glomerularnephritis, infection, lupus nephritis, and renal disease requiring organtransplant; autoimmune disease, such as diabetes mellitus I,inflammatory bowel disease, Crohn's disease, multiple sclerosis,myasthenia gravis, rheumatoid arthritis, and systemic lupuserythematosus; ischemia/reperfusion injury; heart disease, such asmyocardial infarction and cardiac arrest; pregnancy, includingpreeclampsia and fetal hypoxia syndrome; ocular disease, such asage-related macular degeneration, dry eye syndrome, and ocularinfection; organ transplant, including transplant rejection, detectingimminent rejection, detecting infection, and monitoring adjustments inimmunosuppressive drug regimens; infection, including sepsis, pneumonia,bladder infection, urinary tract infection, and kidney infection; andneurological disorders, including multiple sclerosis, Alzheimer'sdisease, Parkinson's disease, schizophrenia, and post-traumatic stressdisorder.

“Control” refers to a sample having a known reference level ofcomplement activation. In some embodiments, the control has a complementactivation level comparable to that of an individual who is notexperiencing a complement-associated disorder, such that a test samplehaving a complement activation level that is deviated compared to thecontrol is indicative of a complement-associated disorder. In certainembodiments, a complement-associated disorder is indicated when the testsample complement activation level is statistically significantlydeviated compared to the control.

“C3 activation signature,” as used herein, means changes in C3activation levels over time.

“Deviated” and “a deviation” as used herein, refer to statisticallysignificant deviations as compared to a reference level in a control.Depending on the analyte being assayed, a deviated test sample level maybe elevated or decreased relative to the control level.

“Decreased,” as compared to a reference level in a control, meansstatistically significantly decreased. In an acute inflammatoryresponse, intact C3 levels are depleted as C3 is broken down into itsactivation products. In certain embodiments, intact C3 levels areconsidered decreased as compared to a reference level in a control atabout 10%.

“Elevated,” as compared to a reference level in a control, meansstatistically significantly elevated. In an acute inflammatory response,iC3b levels increase as C3 is broken down into its activation products.In certain embodiments, a ratio of iC3b to intact C3 that is elevated ascompared to the normal ratio of 0.005 is indicative of C3 activation.

“Epitope” refers to the minimum portion of a molecule that is recognizedby, and thus determines the immunospecificity of, an antibody that bindsto such epitope. The term is also used herein to refer to the minimumportion of a molecule that is recognized by a non-antibody specificbinding agent. Unless otherwise indicated, it is assumed herein that aspecific binding agent that binds to a complement protein binds to anepitope present and accessible for binding in the native protein, i.e.,the epitope is not a neoepitope.

“Inflammatory distress” or “inflammatory dysfunction” occurs when theinflammatory response fails to resolve or remove the stimuli towardwhich the inflammatory response is directed. In such acute cases, theinflammatory response increases until homeostatic control over theprocess erodes. In one embodiment, a complement activation leveldetermined by the assays and methods disclosed herein correlatesdirectly with the severity of inflammatory distress being experienced byan individual. For example, when iC3b concentration is about 12.5% ofintact C3, the patient's inflammatory distress can be said to be mildlysevere. When iC3b concentration is about 2.5-5% of intact C3, thepatient's inflammatory distress can be said to be moderately severe.When iC3b concentration is over 5% of intact C3, the patient'sinflammatory distress is said to be highly severe. Understanding theseverity of a patient's inflammatory distress can inform a physician'streatment of the individual. For example, if the individual presentswith a highly severe inflammatory distress level, as indicated by theassays and methods disclosed herein, the physician can provide emergencymedical treatment within the earliest time points of inflammatorydistress, in order to minimize damage from inflammatory response.

“Label” refers to a moiety that facilitates the direct or indirectdetection and/or quantitative or relative measurement of a molecule towhich it is attached. A detectable label often produces a signal such asfluorescence, chemiluminescence, radioactivity, color, magnetic orparamagnetic properties, etc., that renders it detectable, e.g., by theuse of instruments that detect fluorescence, chemiluminescence,radioactivity, color, magnetic field, magnetic resonance, etc., or insome cases by visual inspection. The label may be, e.g., fluorescentsubstance; pigment; chemiluminescent or luminescent substance; coloredsubstance; magnetic substance; or a non-magnetic metal particle such asgold colloid. In a specific embodiment, the detecting antibodiessuitable for use in the instant methods and assays are conjugated to acolloidal gold label, which provides a color signal.

“Neoepitope” refers to an epitope that is generated or becomesdetectable as a result of proteolytic cleavage of a complement componentor cleavage product.

In certain embodiments of the assays and methods disclosed herein, thecomplement present in the body fluid sample tested is not substantiallyactivated by the assay or method itself. “Not substantially activated,”as used in this context, means that the methods and assays of thepresent invention are substantially free of in vitro activation causedby the test methods and/or materials. In this way, false positive testresults for complement activation are avoided, since the lateral flowimmunoassay is rapid and requires less sample manipulation, thusavoiding many of the stimuli that contribute to in vitro complementactivation.

“Point-of-care,” as used herein, refers to a device or method that canbe used or carried out at the bedside or site of injury of the patient.Point-of-care tests generally do not require shipping a sample to alaboratory for processing or the expertise of a skilled laboratorytechnician. The point-of-care methods and tests described herein allow aclinician to receive critical information at the patient's bedside, orat the site of traumatic injury or triage, which can direct patient careduring the critical first moments after a physiological crisis thattriggers complement activation.

“Reader” refers to an instrument suitable for the detecting of thesignal produced by the label. Various instruments are known in the artfor the detection of label signals in diagnostic testing. In a specificembodiment of the present invention, the label is colloidal gold and thereader is an instrument suitable for the qualitative and/or quantitativedetection of the color signal produced by the label. Suitable readersare available commercially from a variety of vendors, including BioAssayWorks (Ijamsville, Md.), the ESE-Quant from Qiagen (Hilden, Germany),Easterline LRE (Nordlingen, Germany), and Detekt Biomedical (Austin,Tex.). In some specific embodiments, the reader is a handheld readerthat quantifies the amount or concentration of intact C3, iC3b, or totalC3.

“Treatment,” as used herein, encompasses any diagnostic, therapeutic,preventive, or remedial treatment administered to an individual. In someembodiments, treatment encompasses performing additional diagnostictesting on the individual. In other embodiments, treatment encompassestherapeutic treatment, such as administering a therapeutic agent to theindividual. In certain embodiments, the therapeutic agent is selectedfrom the group consisting of antibiotics, anti-inflammatory agents, andinhibitors of complement. In other embodiments, treatment encompassesmodifying a treatment the individual has already received or isreceiving. For example, in one embodiment treating an individual on aventilator encompasses optimizing the ventilator.

Overview of the Complement System

The complement system comprises more than 30 serum and cellular proteinsand plays important roles in innate and adaptive immunity. There arethree major pathways of complement activation. The classical pathway isprimarily activated by immune complexes, specifically IgG/IgM antibodiesbound to antigen. Other activators include lipopolysaccharide, myelin,polyanionic compounds, Creactive protein (CRP), and microbial DNA andRNA. The lectin pathway is activated by polysaccharides withfree-mannose group and other sugars common to fungi and bacteria. Thealternative pathway is mediated by direct C3 activation by “foreign”substances that often include microbial cell wall components. All threemajor pathways of complement activation converge on the central proteincomplement component 3 (C3). C3 is a central mediator of inflammationand is activated by most factors that cause inflammation. See FIGS. 1and 2 for a schematic overview of the complement system.

The classical pathway is typically triggered by immune complexes, whichare complexes of antigen bound with antibodies, generally belonging tothe IgM or IgG isotypes. Immune complexes in turn bind to complementcomponent C1, which is comprised of C1q, C1r, and C1s. The binding ofC1q to an antibody-antigen complex triggers activation of C1r and C1s.Activated C1s then cleaves component C4 to produce C4a and C4b. C4b iscapable of covalent attachment to cell surfaces, although only aboutfive percent does so. The remaining 95 percent reacts with water to forma soluble, activated C4b. Component 2 can then associate with C4b, whichafter which it is activated by C1s to C2a and C2b. C4b and C2a combineto form C4bC2a, the classical pathway (CP) C3 convertase.

The CP convertase cleaves C3 to form C3a and C3b. Like activated C4b,C3b can covalently bind to cell surfaces or react with H₂O and stay insolution. Activated C3b has multiple roles. By itself, it can serve asan opsonin to make the decorated cell or particle more easily ingestedby phagocytes. In addition, C3b can associate with C4bC2a (the CP C3convertase) to form a C5 convertase. The complex, termed C4bC2aC3b istermed the CP C5 convertase. Alternatively, C3b can form the core ofanother C3 convertase called the alternative pathway (AP) C3 convertase.

The alternative pathway (AP) is another mechanism by which C3 can becomeactivated. It is typically activated by targets such as microbialsurfaces and various complex polysaccharides and other materials. Thisalternative pathway can also be initiated spontaneously by the cleavageof the thioester bond in C3 by a water molecule to form C3(H₂O). C3(H₂O)binds factor B, which allows factor D to cleave factor B to Ba and Bb.Bb remains associated with C3(H₂O) to form C3(H₂O)Bb complex, which actsas a C3 convertase and cleaves C3, resulting in C3a and C3b.

C3b formed either via this process or via the classical or lectinpathways binds to targets (e.g., on cell surfaces) and forms a complexwith factor B, which is subsequently cleaved by factor D and form Bb,resulting in C3bBb, which is termed the alternative pathway (AP) C3convertase. Binding of another molecule of C3b to the AP C3 convertaseproduces C3bBbC3b, which is the AP C5 convertase.

The lectin complement pathway is initiated by binding of mannose-bindinglectin (MBL) and MBL-associated serine protease (MASP) to carbohydrates.The MB11 gene (known as LMAN1 in humans) encodes a type 1 integralmembrane protein localized in the intermediate region between theendoplasmic reticulum and the Golgi. The MBL2 gene encodes the solublemannose-binding protein found in serum. In the human lectin pathway,MASp1 and MASP2 are involved in proteolysis of C4 and C2, leading to C3convertase, which lead to production of a C5 convertase as describedabove for the CP.

C5 convertase generated via any of the three pathways cleave C5 toproduce C5a and C5b. C5b then binds to C6, C7, and C8, which catalysespolymerization of C9 to form the C5b-9 membrane attack complex (MAC).The assembling MAC inserts itself into target cell membrane, forming apore delineated by a ring of C9 molecules. MAC formation causes celllysis of invading microbes, MAC formation on host cells can also causelysis, but not necessarily. Sublytic amounts of MAC on the membrane ofcells may affect cell function in a variety of ways. The small cleavageproducts C3a, C4a, and C5a are anaphylatoxins and mediate multiplereactions in the acute inflammatory response. C3a and C5a are alsopotent chemotactic factors that attract immune system cells such asneutrophils and macrophages into the area of crisis.

Complement as a Biomarker for Physiological Crisis

Complement component C3 is useful as a general alert biomarker that thebody is responding to some form of physiological crisis, such as injury,infection, or other disease process. Complement has been associated witha wide variety of diseases, including lupus, arthritis, intracranialhemorrhage, diabetes, multiple sclerosis, heart disease, and age-relatedmacular degeneration. In many cases, the severity of disease correlateswith the level of complement activation. In some cases, complement canplay a role in disease pathology. In these cases, the body is not ableto successfully control the cause of inflammation, which goes from localto systemic. Complement activation can directly damage tissue or do soindirectly by over-activating cells and recruiting immune cells that inturn cause tissue destruction. Examples of over activation includeanaphylactic shock, multiple organ failure (MOF), acute respiratorydistress syndrome (ARDS), and systemic inflammatory response syndrome(SIRS).

Complement activation in the immediate and early post-trauma period hasbeen well documented and occurs by several different mechanisms, likelyinvolving all three major pathways. Release and activation ofproteolytic enzymes may directly activate complement components. Tissuedamage and disruption of the endothelial lining expose surfaces thatlack the endogenous complement inhibiting molecules that normallyprotect host tissues. These surfaces are susceptible to deposition ofC3b and alternative pathway activation. Complement activation is alsotriggered by reperfusion of tissues following post-traumatic ischemia.

Multiple lines of evidence suggest that complement activation is animportant factor in many of the complications of severe trauma,contributing significantly to 1/R injury, ARDS, MODS, secondary CNSinjury, and sepsis. First, it is clear that complement activation is acommon occurrence in the immediate post-trauma period in human traumavictims, and several studies have provided evidence suggesting that theextent of complement activation correlates positively with pooroutcomes. Second, there is considerable evidence that complementactivation is a major cause of I/R injury in animal models of trauma aswell as in human trauma victims. Third, numerous studies havedemonstrated that complement deficiency or administration of complementinhibitors reduces tissue damage and improves outcomes in a variety ofexperimental models including hemorrhage, I/R injury, and CNS injury.

Several studies measured complement activation in trauma patients atsequential time points following severe trauma and investigated theexistence of a correlation between complement activation and injuryseverity. Adverse outcomes such as ARDS, multi-organ failure, sepsis,and death were also monitored in relation to complement activation. Inone study, complement parameters were determined over 14 days in traumapatients at risk of ARDS. All patients showed a decrease in serum levelsof C3, C4, C5 and of the inhibitors C1-INH, complement factor H (CFH),and complement factor I (CFI) in the first 24 hours, indicatingconsumption by high levels of complement activation. See Catania et al.,Immunological consequences of trauma and shock, Ann. Acad. Med.Singapore 28:120-32 (1999); Hecke, et al., Circulating complementproteins in multiple trauma patients—correlation with injury severity,development of sepsis, and outcome, Crit. Care Med. 25(12): 201524(1997); Huber-Lang et al., Complement-induced impairment of innateimmunity during sepsis, J, Immunol. 169:3223-31 (2002); Kang et al.,Change of complement system predicts the outcome of patients with severethermal injury, J. Burn Care Rehabil. 24:148-53 (2003); and Younger etal., Detrimental effects of complement activation in hemorrhagic shock,J. Appl. Physiol. 90:441-46 (2001).

Assays for the Detection and Quantification of Complement Activation andtheir Methods of Use

Presently disclosed assays and methods provide several advantages overprevious complement assays and methods known in the art: for example,the instant assays and methods are suitable for point-of-care use,producing results in a matter of minutes, rather than hours. The rapidreturn of results allows a clinician to act upon changes in C3activation in near real-time to direct patient care during the criticalfirst moments after traumatic injury or at the onset of physiologiccrisis. Provided assays and methods are relatively easy to use and donot require the availability of an outside laboratory or a skilled labtechnician. Alternatively or additionally, provided assays and methodsrequire fewer handling steps, and thus minimize intact C3 activation dueto handling and processing, which leads to false positive test results.Alternatively or additionally, provided assays and methods describedherein employ antibody pairs carefully selected to allow for measurementof the complement proteins intact C3 and/or iC3b, C3's major activationbiomarker. This more precise measurement of complement activation, incomparison to traditional assays of total C3, permits analysis ofturnover and actual amount of C3 remaining and available for activation.

One aspect of the invention encompasses a method including steps ofdetecting in a sample from a subject a level of intact C3, wherein thedetecting involves specific interaction between the intact C3 and anon-cross-reactive antibody thereto, comparing the detected level with areference level, which reference level is within a range of about 350ug/ml to about 1,700 ug/ml, wherein determination that the detectedlevel is below the reference level indicates that the subject issuffering from or susceptible to undesirable and/or pathologiccomplement activation, and administering treatment to treat undesiredcomplement activation if the detected level is below the referencelevel.

Samples used in methods of the present invention may vary according tothe specific application of the invention. Typically, a sample will betaken from an individual suffering from a complement-associateddisorder. Exemplary complement-associated disorders include trauma,inflammatory distress, autoimmune disorders, intracranial hemorrhage,infection such as bacteremia, transplant rejection, ocular disease,heart disease, ischemia/reperfusion injury, age-related maculardegeneration, paroxysmal noctural hemoglobinuria (PNH), hereditaryangiodema, renal disease, pregnancy-associated disorders, andneurological disorders. In some embodiments, the complement-associateddisorder is an autoimmune disorder. Autoimmune disorders include avariety of diseases and conditions associated with an inappropriateimmune response against tissues and substances normally found in thebody. Examples of autoimmune diseases include, but are not limited to,systemic lupus erythematosus, amyotrophic lateral sclerosis, Celiacdisease, Crohn's disease, Graves' disease, and rheumatoid arthritis,among others. In other embodiments, the complement-associated disorderis inflammatory distress. Inflammatory distress, also known asinflammatory dysfunction, includes a variety of diseases and conditionsassociated with hyperinflammation. Examples of diseases and conditionsassociated with inflammatory distress include, but are not limited to,organ failure, systemic inflammatory response syndrome (SIRS), adultrespiratory distress syndrome (ARDS), sepsis, ventilator associatedpneumonia (VAP), respiratory distress and pneumonia.

According to several aspects of the invention, the sample may be asample of body fluid or derived therefrom. Exemplary body fluids thatmay comprise or be processed to produce a sample include whole blood,serum, plasma, urine, tears, saliva, wound exudate,broncheoalveolar-lavage fluid, and cerebrospinal fluid. See FIG. 19 fora non-limiting list of suitable body fluids. In some embodiments, thebody fluid may be obtained from the individual within one hour of aphysiological event triggering complement activation. In otherembodiments, the body fluid may be whole blood.

Complement activation levels may be assessed for deviation from areference value of a control (i.e. a “normal” level) which indicatescomplement is activated in the individual. As an example, in certainembodiments, the level or concentration of iC3b in the test sample maybe elevated in comparison to a control, indicating C3 is activated andhas been further split into its activation product, iC3b. As a furtherexample in other embodiments, the level or concentration of intact C3 isdecreased in comparison to a control, indicating intact C3 has beenconverted to its breakdown or activation products and is hence depletedin the individual.

In some embodiments, a “normal” intact C3 level in a patient sample(e.g., bodily fluid) falls within a range with a lower boundary and anupper boundary that is higher than the lower boundary. In someembodiments, the lower boundary is selected from the group consisting of30 ug/ml, 35 ug/ml, 40 ug/ml, 45 ug/ml, 50 ug/ml, 55 ug/ml, 60 ug/ml, 65ug/ml, 70 ug/ml, 75 ug/ml, 80 ug/ml, 85 ug/ml, 90 ug/ml, 95 ug/ml, 100ug/ml, 110 ug/ml, 120 ug/ml, 130 ug/ml, 140 ug/ml, 150 ug/ml, 160 ug/ml,170 ug/ml, 180 ug/ml, 190 ug/ml, 200 ug/ml, 210 ug/ml, 220 ug/ml, 230ug/ml, 240 ug/ml, 250 ug/ml, 260 ug/ml, 270 ug/ml, 280 ug/ml, 290 ug/ml,300 ug/ml, 350 ug/ml, 400 ug/ml, 450 ug/ml, 500 ug/ml, 550 ug/ml, 600ug/ml 650 ug/ml, 700 ug/ml or more. In some embodiments, the upperboundary is selected from the group consisting of 2,000 ug/ml, 1,900ug/ml, 1,800 ug/ml, 1,700 ug/ml, 1,600 ug/ml, 1,500 ug/ml, 1,400 ug/ml,1,300 ug/ml, 1,200 ug/ml, 1,100 ug/ml, 1,000 ug/ml, 900 ug/ml, 800ug/ml, 700 ug/ml, 600 ug/ml, 500 ug/ml, 400 ug/ml, 300 ug/ml or less. Insome embodiments a “normal” level of intact C3 falls within the range of30 ug/ml-2,000 ug/ml; in other embodiments a “normal” level of intact C3falls within the range of 100 ug/ml-1,500 ug/ml; in other embodiments a“normal” level of intact C3 falls within the range of 200 ug/ml-1,200ug/ml; in other embodiments a “normal” level of intact C3 falls withinthe range of 200 ug/ml-1,000 ug/ml; in other embodiments a “normal”level of intact C3 falls within the range of 200 ug/ml-800 ug/ml; inother embodiments a “normal” level of intact C3 falls within the rangeof 200 ug/ml-500 ug/ml; in other embodiments a “normal” level of intactC3 falls within the range of 600 ug/ml-1,800 ug/ml; in other embodimentsa “normal” level of intact C3 falls within the range of 700 ug/ml-1,700ug/ml.

In some embodiments, where the sample is blood, plasma or serum, a“normal” level of intact C3 may fall within the range 300 ug/ml-1,700ug/ml; in other embodiments, a “normal” level of intact C3 may fallwithin the range 400 ug/ml-1,400 ug/ml; in still other embodiments, a“normal” level of intact C3 may fall within the range 500 ug/ml-1,300ug/ml; in still other embodiments, a “normal” level of intact C3 mayfall within the range 500 ug/ml-1,200 ug/ml; in still other embodiments,a “normal” level of intact C3 may fall within the range 500 ug/ml-1,100ug/ml; in still other embodiments, a “normal” level of intact C3 mayfall within the range 500 ug/ml-1,000 ug/ml; in still other embodiments,a “normal” level of intact C3 may fall within the range 500 ug/ml-900ug/ml; in yet other embodiments, a “normal” level of intact C3 may fallwithin the range 500 ug/ml-800 ug/ml; in yet other embodiments, a“normal” level of intact C3 may fall within the range 500 ug/ml-700ug/ml; in yet other embodiments, a “normal” level of intact C3 may fallwithin the range 500 ug/ml-600 ug/ml.

As a more specific example, in some embodiments where the sample iswhole blood, a “normal” level of intact C3 may fall within the range 500ug/ml-1,000 ug/ml.

As a more specific example, in some embodiments where the sample isplasma, a “normal” level of intact C3 may fall within the range 700ug/ml-1,700 ug/ml.

As a more specific example, in some embodiments where the sample isserum, a “normal” level of intact C3 may fall within the range 700ug/ml-1,700 ug/ml.

In other embodiments, where the sample is tears, a “normal” level ofintact C3 may fall within the range 30 ug-ml-100 ug/ml, in otherembodiments, a “normal” level of intact C3 may fall within the range 40ug/ml-90 ug/ml; in still other embodiments, a “normal” level of intactC3 may fall within the range 50 ug/ml-80 ug/ml; in still otherembodiments, a “normal” level of intact C3 may fall within the range 50ug/ml-70 ug/ml; in still other embodiments, a “normal” level of intactC3 may fall within the range 50 ug/ml-60 ug/ml.

In some embodiments, intact C3 is detected using a non-cross reactiveantibody characterized in that a 1 ug/ul solution of iC3b producessignal equivalent to less than about 1 ng/ml of C3. In otherembodiments, the non-cross reactive antibody is HM2075.

Complement activation level may correlate to a severity of inflammatorydistress: the higher the complement activation level, the greater therisk of developing inflammatory distress and/or the greater the severityof inflammatory distress experienced by the individual. Therefore,according to some embodiments of the invention, thecomplement-associated disorder is inflammatory distress and theconcentration of one or more of intact C3 and iC3b correlates to aseverity of inflammatory distress.

The complement activation level determined by the instant method mayprovide point-of-care diagnostic information that can direct patientcare. Based on the risk of complement-associated disorder or severity ofdisease or disorder, a clinician can select the appropriate treatmentfor the individual. In some embodiments, the treatment comprisesperforming additional testing on the individual to determine the causeof inflammatory distress. For example, severe trauma patients thatrequire ventilator assistance for breathing are at risk for acuterespiratory distress caused either by Ventilator Associated Pneumonia(VAP) or non-infectious inflammatory dysfunction. A level of complementactivation may indicate active or imminent inflammatory dysfunctionbefore clinical signs of respiratory crisis are presented. The instantassays and methods may indicate whether the individual is experiencingVAP or non-infectious respiratory distress. Alternatively, the instantassays and methods may indicate additional testing (such asbroncheoalveolar lavage (BAL)) at a time point earlier than is nowstandard practice. If the individual is suffering from VAP, thetreatment may comprise administering a therapeutic agent such as anantibiotic or set of antibiotics. If the inflammatory dysfunction iscaused by non-infectious means, a therapy may be selected from the groupconsisting of ventilator adjustment, anti-inflammatory agents, andinhibitors of complement.

If the individual is suffering from systemic lupus erythematosus, theadditional testing may be genetic testing. If the individual issuffering from traumatic brain injury or intracranial hemorrhage, theadditional testing may comprise obtaining a cerebrospinal fluid samplefor additional analysis. If the individual is suffering from a wound,including a non-healing wound, the further testing may compriseobtaining a sample of wound exudate for additional analysis.

Many inhibitors of complement are known in the art and suitable for usewith the methods of the present invention. In some embodiments, theinhibitor of complement is selected from the group consisting of naturalcomplement inhibitors and derivatives thereof, compstatin and analogsthereof, anti-membrane attack complex (MAC) antibodies, anti-C3antibodies, anti-C5 antibodies, C3a receptor antagonists, and C5areceptor antagonists. Examples of additional complement inhibitors canbe found, for example, in Emlen et al., Therapeutic complementinhibition: new developments, Semin. Thromb. Hemost. 36(6):660-68(2101); Wagner et al., Therapeutic potential of complement modulation,Nat. Rev. Drug Discov. 9(1):43-56 (21010); and Ricklin et al.,Complement-targeted therapeutics, Nat. Biotechnol. 25(11):1265-75(2007), the contents of which are incorporated by reference herein intheir entirety.

One of the benefits of methods of the present invention is the rapidreturn of results, which enables a clinician to direct patient care inresponse to changes in complement activation in near real-time. Whereasprevious assays for complement activation known in the art require fulllaboratories, skilled technicians, and hours to complete, the instantmethods and assays provide results in a much shorter time frame. In someembodiments, the instant method may provide a measurement of thecomplement activation level in the sample in about 30 minutes or less.In other embodiments, the method may provide a complement activationlevel in the sample in about 30, about 25, about 20, about 15, about 10,about 5, or about 3 minutes or less. The rapidity of the method enablesthe clinician to determine a complement activation level and select anappropriate therapy in response, during a clinically-meaningful timeperiod. Indeed, the instant methods can be carried out at the bedside oreven at the site of traumatic injury—for example, in an ambulance or intriaging a patient on the battlefield—and the complement activationlevel determined by the assay and method can direct patient care withinthe critical first hour post-trauma.

Another aspect of the invention encompasses a method including the stepsof detecting in a sample from a subject a level of iC3b wherein thedetecting involves specific interaction between the iC3b and anon-cross-reactive antibody thereto, comparing the detected level with areference level, which reference level is within a range of about 10ng/ml to about 5,000 ng/ml, wherein determination that the detectedlevel is above the reference level indicates that the subject issuffering from or susceptible to undesirable and/or pathologiccomplement activation, and administering treatment to treat undesiredcomplement activation if the detected level is above the reference level

Samples used in methods of the present invention may from any of avariety of sources. Typically, a sample will be taken from an individualsuffering from a complement-associated disorder. Exemplarycomplement-associated disorders include trauma, inflammatory distress,autoimmune disorders, intracranial hemorrhage, infection such asbacteremia, transplant rejection, ocular disease, heart disease,ischemia/reperfusion injury, age-related macular degeneration,paroxysmal noctural hemoglobinuria (PNH), hereditary angiodema, renaldisease, pregnancy-associated disorders, and neurological disorders. Insome embodiments, the complement-associated disorder is an autoimmunedisorder. Autoimmune disorders include a variety of diseases andconditions associated with an inappropriate immune response againsttissues and substances normally found in the body. Examples ofautoimmune diseases include, but are not limited to, systemic lupuserythematosus, amyotrophic lateral sclerosis, Celiac disease, Crohn'sdisease, Graves' disease, and rheumatoid arthritis, among others. Inother embodiments, the complement-associated disorder is inflammatorydistress. Inflammatory distress, also known as inflammatory dysfunction,includes a variety of diseases and conditions associated withhyperinflammation. Examples of diseases and conditions associated withinflammatory distress include, but are not limited to, organ failure,systemic inflammatory response syndrome (SIRS), adult respiratorydistress syndrome (ARDS), sepsis, ventilator associated pneumonia (VAP),respiratory distress and pneumonia.

According to several aspects of the invention, the sample may be asample of body fluid or derived therefrom. Exemplary body fluids thatmay comprise or be processed to produce a sample include whole blood,serum, plasma, urine, tears, saliva, wound exudate, broncheoalveolarlavage fluid, and cerebrospinal fluid. See FIG. 19 for a non-limitinglist of suitable body fluids. In some embodiments, the body fluid may beobtained from the individual within one hour of a physiological eventtriggering complement activation. In other embodiments, the body fluidmay be whole blood.

In some embodiments, a “normal” iC3b level in a patient sample (e.g.,bodily fluid) falls within a range with a lower boundary and an upperboundary that is higher than the lower boundary. In some embodiments,the lower boundary is selected from the group consisting of 75 ng/ml, 80ng/ml, 85 ng/ml, 90 ng/ml, 95 ng/ml, 100 ng/ml, 110 ng/ml, 120 ng/ml,130 ng/ml, 140 ng/ml, 150 ng/ml, 160 ng/ml, 170 ng/ml, 180 ng/ml, 190ng/ml, 200 ng/ml, 210 ng/ml, 220 ng/ml, 230 ng/ml, 240 ng/ml, 250 ng/ml,260 ng/ml, 270 ng/ml, 280 ng/ml, 290 ng/ml, 300 ng/ml or more. In someembodiments, the upper boundary is selected from the group consisting of5 ug/ml, 4.5 ug/ml, 4 ug/ml, 3.5 ug/ml, 3 ug/ml, 2.5 ug/ml, 2 ug/ml, 1.9ug/ml, 1.8 ug/ml, 1.7 ug/ml, 1.6 ug/ml, 1.5 ug/ml, 1.4 ug/ml, 1.3 ug/ml,1.2 ug/ml, 1.1 ug/ml, 1.0 ug/ml, 0.9 ug/ml, 0.8 ug/ml, 0.7 ug/ml, 0.6ug/ml, 0.5 ug/ml, 0.4 ug/ml, 0.3 ug/ml or less. In some embodiments a“normal” level of iC3b falls within the range of 100 ng/ml-5 ug/ml; inother embodiments a “normal” level of iC3b falls within the range of 100ng/ml-4 ug/ml; in other embodiments a “normal” level of iC3b fallswithin the range of 100 ng/ml-3 ug/ml; in other embodiments a “normal”level of iC3b falls within the range of 100 ng/ml-2 ug/ml; in otherembodiments a “normal” level of iC3b falls within the range of 100ng/ml-1.5 ug/ml; in other embodiments a “normal” level of iC3b fallswithin the range of 200 ng/ml-1 ug/ml; in other embodiments a “normal”level of iC3b falls within the range of 200 ng/ml-0.7 ug/ml; in otherembodiments a “normal” level of iC3b falls within the range of 200ng/ml-0.5 ug/ml; in other embodiments a “normal” level of iC3b fallswithin the range of 200 ng/ml-0.3 ug/ml.

In some embodiments, where the sample is blood, plasma or serum, a“normal” level of iC3b may fall within the range 150 ng/ml-5,000 ng/ml;in other embodiments a “normal” level of iC3b may fall within the range150 ng/ml-4,000 ng/ml; in other embodiments a “normal” level of iC3b mayfall within the range 150 ng/ml-3,000 ng/ml; in other embodiments a“normal” level of iC3b may fall within the range 150 ng/ml-2,000 ng/ml;in other embodiments a “normal” level of iC3b may fall within the range150 ng/ml-1,000 ng/ml; in other embodiments a “normal” level of iC3b mayfall within the range 175 ng/ml-900 ng/ml; in still other embodiments a“normal” level of iC3b may fall within the range 200 ng/ml-800 ng/ml; instill other embodiments a “normal” level of iC3b may fall within therange 200 ng/ml-700 ng/ml; in still other embodiments a “normal” levelof iC3b may fall within the range 200 ng/ml-600 ng/ml; in still otherembodiments a “normal” level of iC3b may fall within the range 200ng/ml-500 ng/ml; in still other embodiments a “normal” level of iC3b mayfall within the range 200 ng/ml-400 ng/ml; in still other embodiments a“normal” level of iC3b may fall within the range 200 ng/ml-300 ng/ml.

As a more specific example, in some embodiments where the sample iswhole blood, a “normal” level of iC3b may fall within the range 10ng/ml-1,500 ng/ml.

As a more specific example, in some embodiments where the sample isplasma, a “normal” level of iC3b may fall within the range 10ng/ml-3,000 ng/ml.

As a more specific example, in some embodiments where the sample isserum, a “normal” level of iC3b may fall within the range 10 ng/ml-5,000ng/ml.

In some embodiments, where the sample is tears, a “normal” level of iC3bmay fall within the range 1 ng-ml-50 ng/ml; in other embodiments, a“normal” level of iC3b may fall within the range 1 ng/ml-40 ng/ml; instill other embodiments, a “normal” level of iC3b may fall within therange 1 ng/ml-30 ng/ml; in yet other embodiments, a “normal” level ofiC3b may fall within the range 1 ng/ml-20 ng/ml; in still otherembodiments, a “normal” level of iC3b may fall within the range 2ng/ml-10 ng/ml; in yet other embodiments, a “normal” level of iC3b mayfall within the range 4 ng/ml-10 ng/ml.

In some embodiments, iC3b is detected using a non-cross reactiveantibody characterized in that a 1 ug/ul solution of intact C3 producessignal equivalent to less than about 1 ng/ml of iC3b. In otherembodiments, the non-cross reactive antibody is selected from the groupconsisting of A209, MCA2607, and HM2199.

In still another aspect, the invention encompasses a method includingthe steps of detecting in a sample a ratio of intact C3 level to iC3blevel, wherein the detecting involves specific interaction between theintact C3, the iC3b, or both with a non-cross-reactive antibody thereto,comparing the detected level with a reference ratio of about 0.001wherein determination that the detected level is below the referencelevel indicates that the subject is suffering from or susceptible toundesirable and/or pathologic complement activation, and administeringtreatment to treat undesired complement activation if the detected levelis below the reference level.

Some embodiments of the invention will comprise a reference ratio in therange of 0.001 to 0.005. In certain embodiments, the reference ratio isselected from the group consisting of 0.001, 0.002, and 0.005.

In some embodiments methods of the present invention, the form of theassay may be a lateral flow immunoassay that detects the presence orabsence of one or more of intact C3 and iC3b in the sample. In otherembodiments of the method, the lateral flow assay detects the presenceof total C3. In still other embodiments, the lateral flow immunoassay isread by a reader. In a more specific embodiment, the reader quantifies aconcentration of one or more of intact C3 and iC3b in the sample. Inanother specific embodiment, the reader quantifies a concentration oftotal C3 in the sample.

In lateral flow assay embodiments of the invention, U.S. Pat. No.7,910,381 (Ford et al.) describes several such assays that arecontemplated as useful in accordance with the present invention and thedisclosure of this patent is hereby incorporated in its entirety.Embodiments of the disclosed lateral flow assays are presently soldunder the CFLAT® name. FIG. 20 shows one embodiment of a lateral flowassay cassette in accordance with the teachings of U.S. Pat. No.7,910,381.

In certain embodiments, the clinician may detect a decrease incomplement activation in response to the treatment the patient isreceiving. Accordingly, the clinician may then modify the individual'streatment by adjusting the dosing of medications administered, such asanti-inflammatory agents or complement inhibitors, or discontinuingtreatment once complement levels have returned to normal (i.e., a levelin an individual who is not experiencing a complement-associateddisorder). In other embodiments, the clinician may detect a rise incomplement activation levels in response to the treatment the patient isreceiving. Accordingly, the clinician may then modify the individual'streatment by increasing the dosage of medications, such asanti-inflammatory agents or complement inhibitors, until a desiredstabilization or decrease in complement activation levels is achieved.If no change in complement activation level is detected, the clinicianmay modify the individual's treatment or may elect to maintain theindividual's treatment regimen until a change in complement activationlevels is observed.

Referring to FIG. 4, a lateral flow immunoassay embodiment of thepresent invention is described herein and is comprised of a cellulosemembrane strip 3, upon which is disposed a sample pad 1 to absorb thesample fluid and allow gradual migration of thesample-and-particle-conjugate immune complexes, a wick 6 at the distalend of the strip that absorbs the liquid sample and conjugate materialto facilitate capillary migration through the cellulose membrane strip3, and a particle conjugate pad 2 comprising a detecting antibody boundto a label, or detection conjugate. The cellulose membrane strip 3 isthe test zone region, upon which is disposed a test line 4, comprisingmonoclonal or polyclonal antibodies striped for capturing the detectionconjugate and a control line 5, comprising an antibody that binds acontrol analyte, such as IgG, and indicates to the user that the testwas successfully run. The lateral flow immunoassay further comprises apolyester film backing 7 attached to the cellulose membrane strip 3, anda pressure-sensitive laminate film backing 8. Each lateral flowimmunoassay may be packaged in a MYLAR® zero-vapor barrier pouch, forexample.

When a test sample is applied to the sample pad 1, the sample migratesfrom the sample pad 1 through the particle conjugate pad 2, where anytarget analyte present will bind to the detecting antibody conjugate.The sample then continues to migrate across the membrane 3 until itreaches the test line 4 where the target/conjugate complex will bind tothe immobilized antibodies producing a visible line on the membrane. Thesample then migrates further along the membrane strip 3 until it reachesthe control line 5, where excess antibody conjugate that did not bindthe test line will bind the control line and produce a second visibleline on the membrane. The control line ligand is often an antibodyagainst the Fe region of the conjugated antibody. This control lineindicates that the sample has migrated across the membrane as intended.

In certain embodiments, a lateral flow immunoassay in accordance withthe present invention comprises a single membrane strip for thedetection of a single analyte. In other embodiments, the lateral flowimmunoassay detects two or more analytes. When the lateral flowimmunoassay detects two or more analytes, the test can be configuredwith multiple membrane strips arranged in parallel (see, for example,the schematic of FIG. 5(B)), or with multiple test lines arranged inseries on a single membrane strip (see, for example, the schematic ofFIG. 9(B)).

Referring to FIG. 6, in some embodiments, the lateral flow immunoassaymembrane strip is enclosed in a test cassette 9 having a port 10 forinstilling the test sample and a window 11 for viewing the test results.The lateral flow immunoassays of FIG. 6 are configured to assay for asingle analyte and each comprise one test line 4 and one control line 5.

Referring to FIG. 7, in some embodiments, the lateral flow immunoassayis configured to test for two analytes in a single test cassette inparallel. In some embodiments, the lateral flow immunoassay comprisestwo ports 10 for instilling the test samples and a separate membranestrip 3 for each analyte (see FIG. 7(A)). In other embodiments, thelateral flow immunoassay comprises one port 10 for instilling the sampleand a separate membrane strip 3 for each analyte (see FIG. 7(B)).

Referring to FIG. 8, in some embodiments, the lateral flow immunoassaymay be configured to test for three analytes in a single test cassettein parallel. In some embodiments, the lateral flow immunoassay maycomprise three ports 10 for instilling the test sample and a separatemembrane strip 3 for each analyte (see FIG. 8(A)). In other embodiments,the lateral flow immunoassay may comprise one port 10 for instilling thetest sample and a separate membrane strip 3 for each analyte (see FIG.8(B)).

Referring to FIG. 10, in certain embodiments, the lateral flowimmunoassay may be configured to test for multiple analytes in a singletest cassette in series. FIG. 10(A) depicts a test cassette comprising amembrane strip 3 with two test lines 4 and one control line 5 arrangedin series. FIG. 10(B) depicts a test cassette comprising a membranestrip 3 with three test lines 4 and one control line 5 arranged inseries.

The lateral flow immunoassay presently disclosed may provide qualitativeand/or quantitative detection of the target markers. Qualitatively, twoclear lines on the membrane may represent a positive result, whereas asingle line in the control zone may represent a negative result.

In some embodiments of the invention, a lateral flow immunoassay for thepoint-of-care detection of a marker of complement activation in a bodyfluid sample comprising complement proteins is provided, with theexemplary lateral flow immunoassay comprising: a membrane strip; adetecting antibody that binds a first epitope of the marker; a test linecomprising a capturing antibody that binds a second epitope of themarker; and a control line comprising an antibody that binds a controlanalyte, wherein the marker is selected from the group consisting ofintact C3 and iC3b.

An exemplary embodiment of such a device is shown in FIG. 20, with (A)showing a conceptual rendering of the structure of the device with themembrane strip being a nitrocellulose membrane strip and employing agold-labeled conjugate pad, which is desirable in certain applications,and (B) showing a method of using the embodiment shown in (A) to assesscomplement activation levels in a sample.

In some embodiments, the detecting antibody comprises a label thatprovides a signal that can be read visually by a clinician orelectronically via a commercial reader. Various labels are suitable foruse in the instantly disclosed assays. In a specific embodiment, thelabel may be colloidal gold as shown in FIG. 20(A).

According to several embodiments, detecting and capturing antibody pairsshould be carefully selected to avoid interfering crosstalk between C3and iC3b. The primary concern is intact C3 producing a signal in anassay for the detection of iC3b. As both molecules are derived from thesame protein molecule, crosstalk can present a problem. As C3 is presentat levels about 200 times higher than iC3b in normal individuals, even aslight degree of crosstalk can have a major impact on the accuratemeasurement of iC3b and C3 activation. This is further complicated bythe fact that improper handling, improper storage, and even reagentsthemselves can cause in vitro C3 activation. Surprisingly, Applicantsdiscovered that not all antibodies suitable for use in traditional ELISAassays are equally suitable for use in the assays of the instantinvention.

Tables 1 and 2 below show the difficulties in identifying antibody pairssuitable for use in the assays of the instant invention. The inventorsanalyzed 19 pairs of antibodies in the intact C3 immunoassay and 18pairs of antibodies in the iC3b lateral flow immunoassay. Of thesepairs, Hycult® HM2075 and MP Biomedicals® 55237 yielded the bestresults, with no cross-reactivity, in the intact C3 lateral flowimmunoassay. Quidel® A209 with either MP Biomedicals® 55237 or Quidel®A250 yielded the best results in the iC3b lateral flow immunoassay.Interestingly, the inventors noted that antibody pairs suitable for usein traditional ELISA assays are not necessarily equally suitable for usein the lateral flow immunoassays described herein. For example, Hycult®HM2198 yielded an assay with about a 1% cross-reactivity, withconsiderable test-to-test variability. This cross-reactivity produced afalse positive iC3b signal at a level of twice that of normalcirculating iC3b. As actual double or tripling of iC3b levels would besigns of massive complement activation, a lateral flow immunoassay with1% cross reactivity is without clinical utility. MP Biomedicals® (55237)worked far better, producing cross reactivity of less than about 0.5%(about 0.05%), compatible with clinical utility. However, it isnoteworthy that both antibodies performed equally well in traditionalELISA assays.

TABLE 1 Antibody Screening Results in intact C3 assay capturing antibodydetection antibody species antigen supplier species antigen suppliernotes mouse C3a Hycult (HM2075) goat C3 MP Biomedicals (55237) no crossreactivity under assay conditions mouse C3a Quidel (A203) goat C3 MPBiomedicals (55237) assay to assay variance too high chicken C3a GenTex(GTX78198) rabbit C3d Abcam (ab15981) no positive readings (doesn'twork) mouse C3a Quidel (A203) rabbit C3d Abcam (ab15981) cross-reactswith C3b/iC3b+++ goat C3a SantaCruz (sc17237) rabbit C3d Abcam (ab15981)the AB may only react with C3a, not intact C3 mouse C3a Hycult (HM2073)rabbit C3d Abcam (ab15981) cross reacts with C3b/iCb+ mouse C3a Hycult(HM2074) rabbit C3d Abcam (ab15981) no positive readings chicken C3aAbcam (ab48580) rabbit C3d Abcam (ab15981) no positive readings mouseC3a Hycult (HM2073) chicken C3a Abcam (ab48580) no positive readingsmouse C3a Quidel (A203) chicken C3a Abcam (ab48580) no positive readingschicken C3a Abcam (ab48580) goat C3 MP Biomedicals (55237) cross reactswith C3b/iC3b+++ mouse C3a Hycult (HM2073) goat C3 MP Biomedicals(55237) cross reacts with C3b/iC3b+++ goat C3a SantaCruz (sc17237) goatC3 MP Biomedicals (55237) similar to HM2073, better in diluted serumrabbit C3d Abcam(ab15981) mouse C3a Quidel (A203) when anti-C3d iscapture Ab, it binds to C3b and iC3b rabbit C3d Abcam(ab15981) chickenC3a GenTex (GTX78198) as well, which prevents efficient binding ofintact C3 rabbit C3d Abcam (ab15981) goat C3a SantaCruz (sc17237) whenanalyzing mixed samples. rabbit C3d Abcam (ab15981) chicken C3a Abcam(ab48580) rabbit C3d Abcam (ab15981) mouse C3a Hycult (HM2073) rabbitC3d Abcam (ab15981) mouse C3a Hycult (HM2074)

TABLE 2 Antibody Screening Results in iC3b assay capturing antibodydetection antibody species antigen supplier species antigen suppliernotes mouse iC3b Quidel (A209) goat C3 MP Biomedicals (55237) no crossreactivity under assay conditions, good signal mouse iC3b AbD serotec(MCA2607) goat C3 MP Biomedicals (55237) cross reacts with C3b/C3c athigh cone mouse iC3b AbD serotec (MCA2607) rabbit C3d Abcam (ab15981)lower signal strength than using anti-C3 mouse iC3b Quidel (A209) rabbitC3d Abcam (ab15981) lower signal strength than using anti-C3 mouse iC3bQuidel (A209) rat C3d Hycult (HM2198) good signal, lower than usinganti-C3 mouse iC3b Quidel (A209) rat C3g Hycult (HM2199) no signal mouseiC3b Quidel (A209) mouse neo Quidel (A250) lower signal strength thanusing HRP-anti C3, C3d better specificity than using anti-C3d rat iC3bHycult (HM2199) goat C3 MP Biomedicals (55237) good signal rat iC3bHycult (HM2199) mouse active Hycult (HM2168) weak signal C3 rat iC3bHycult (HM2199) mouse active Hycult (HM2257) no signal C3 rat iC3bHycult (HM2199) mouse iC3b Quidel (A209) no signal rat iC3b Hycult(HM2199) mouse neo Quidel (A250) no signal C3d mouse active C3 Hycult(HM2168) goat C3 MP Biomedicals (55237) too much crosstalk with C3 mouseactive C3 Hycult (HM2168) rat C3g Hycult (HM2199) weak signal mouseactive C3 Hycult (HM2257) goat C3 MP Biomedicals (55237) no signal mouseactive C3 Hycult (HM2257) rat C3g Hycult (HM2199) no signal mouse C3alpha Meridian (H54189M) goat C3 MP Biomedicals (55237) no signal mouseneo C3d Quidel (A250) rat C3g Hycult (HM2199) very low signal

In one exemplary embodiment, the marker may be intact C3 and thedetecting antibody binds a first epitope of intact C3, wherein the firstepitope is a C3a domain which is present on intact C3 and which is lostupon activation of C3. In a further embodiment, the marker is intact C3and the capturing antibody binds a second epitope on C3, wherein thesecond epitope is a region in the C3d domain which is present on intactC3, C3b, iC3b, and C3d. See FIG. 3(A).

In another exemplary embodiment, the marker may be iC3b and thedetecting antibody binds a first epitope of iC3b, wherein the firstepitope is a neoepitope on iC3b which is revealed when C3b isdeactivated to iC3b and which is occluded when iC3b is further degradedto C3c and C3d. In a further embodiment, the marker is iC3b and thecapturing antibody binds a second epitope on iC3b, wherein the secondepitope is a neoepitope present only on C3b, iC3b, and C3dg. See FIG.3(B).

In a specific example, the marker is intact C3, the capturing antibodyis Hycult® HM2075 and the detecting antibody is MP Biomedicals® 55237.In another very specific example, the marker is iC3b, the capturingantibody is Quidel® A209 and the detecting antibody is MP Biomedicals®55237. In another specific example, the marker is iC3b, the capturingantibody is Quidel® A209 and the detecting antibody is Quidel® A250.

One skilled in the art will appreciate that various control analytes aresuitable for use in the methods of the instant invention to provideverification that the assay was successfully completed. In oneembodiment, the control analyte is IgG.

Another advantage of the instant methods is the avoidance of substantialcomplement activation in the sample by virtue of the test itself, whichcan lead to false positive results. It is well known that C3 is afastidious protein capable of self activation due to sample handling,storage, and contact with foreign materials or substances. Thus, thenature of C3 can lead to false positives in traditional ELISA andturbidity assays for complement activation that involve extensive samplehandling and multiple steps. The instant methods avoid such falsepositives by reducing and/or eliminating sample preparation and handlingsteps, particularly when used in the context of a lateral flow assay,for example. Accordingly, in one embodiment of the methods, complementin the body fluid sample is not substantially activated experimentallyby a lateral flow immunoassay.

In some embodiments of the present methods, it is desirable to have alateral flow immunoassay that can detect more than one marker ofcomplement activation in a single assay. For example, a dual lateralflow immunoassay that can qualitatively and quantitatively detect bothintact C3 and iC3b in the same aliquot of a body fluid may be highlydesirable. Hence, in another embodiment, a lateral flow immunoassay forthe point-of-care detection of markers of complement activation in abody fluid sample comprising complement proteins is provided, thelateral flow immunoassay comprising: a membrane strip; a first detectingantibody that binds a first epitope of intact C3; a first test linecomprising a first capturing antibody that binds a second epitope of theintact C3; a second detecting antibody that binds a first epitope ofiC3b; a second test line comprising a second capturing antibody thatbinds a second epitope of iC3b; and at least one control line comprisingan antibody that binds a control analyte.

In some embodiments, the first and second detecting antibodies comprisea label that provides a signal. Various labels are suitable for use inthe instantly disclosed methods. In some embodiments, the label iscolloidal gold.

According to several aspects of the invention complement in the sampleis not substantially activated experimentally by the lateral flowimmunoassay itself.

In some embodiments, the first detecting antibody binds a first epitopeof intact C3, wherein the first epitope of intact C3 is a C3a domainwhich is present on intact C3 and which is lost upon activation of C3.

In other embodiments, the first capturing antibody binds a secondepitope of intact C3, wherein the second epitope is a region in the C3ddomain which is present on intact C3, C3b, iC3b, and C3d.

In still other embodiments, the second detecting antibody binds a firstepitope of iC3b, wherein the first epitope of iC3b is a neoepitope oniC3b which is revealed when C3b is deactivated to iC3b and which isoccluded when iC3b is further degraded to C3c and C3d.

In yet other embodiments, the second capturing antibody binds a secondepitope of iC3b, wherein the second epitope of iC3b is a neoepitopepresent only on C3b, iC3b, and C3dg.

In still other embodiments, the antibodies that bind intact C3 and theantibodies that bind iC3b are not substantially cross-reactive.

According to several embodiments, the detecting and comparing steps maybe carried out in 30 minutes or less.

EXAMPLES

The following examples are given by way of illustration and are in noway intended to limit the scope of the present invention.

Example 1 Patient Triage

Before the first test sample is assayed, a standard curve is performedusing 10 ng/ml, 30 ng/ml, 100 ng/ml, 300 ng/ml, and 1000 ng/ml of intactC3 and iC3b standards. Lateral flow immunoassay cassettes are read withan electronic reader after 20 minutes.

The test is used to gauge injury severity within 15, 30, or 60 minutesof injury. It is most useful for patients who may have suffered injuriesnot obvious by visual inspection. A drop of blood is collected eitherfrom an arterial line (A-line) or finger stick. A 10 ul sample is drawnup using a fixed volume pipet. The sample then mixed with 990 ul ofsample buffer. The blood and sample buffer are mixed. Using a fixedvolume pipet bulb, 100 ul is drawn up and pipetted onto the lateral flowimmunoassay cassette containing integrated intact C3 and iC3b teststrips. Alternatively, 100 ul can be applied to separate intact C3 andiC3b lateral flow assay cassettes. After 10 minutes but before 40minutes, the cassette is read and results recorded, preferablyelectronically by a reader. If the first reading has an iC3b level (orequivalent iC3b:intact C3 ratio) higher than 50 μg/ml in blood, evidenceof complement activation and high inflammation exist. Staff assumessevere injury and alerts ER staff. Otherwise, a second reading is taken5 minutes later. If the iC3b level (or equivalent iC3b:intact C3 ratio)is higher than 50 μg/ml in blood or the iC3b level has increased by morethan 25%, the patient is assumed to have severe injury and ER staff isalerted. A lesser increase or no increase is suggestive, but notconclusive, of less severe injury.

Example 2 Trajectory Monitoring of a Trauma Patient

At the beginning of the shift, ICU staff performs a standard curve using10 ng/ml, 30 ng/ml, 100 ng/ml, 300 ng/ml, and 1000 ng/ml of intact C3and iC3b standards. Lateral flow immunoassay cassettes are read with anelectronic reader after 20 minutes.

The objective of trajectory monitoring is to detect changes ininflammatory and immune status of patients that have been stabilizedafter severe trauma. In this example, respiratory distress caused byeither pneumonia or inflammatory dysfunction is to be detected. Theexpected patient profile is one who has an injury severity score (ISS)equal or greater to 16 and who requires ventilator assistance forbreathing.

The patient receives a complement test at frequent intervals, whichaligns with the time points for testing glucose levels in blood. Thisinterval between testing is usually about two hours. Blood is collectedusing the same method as for glucose testing, either by A-line or byfinger stick. A 10 ul sample is drawn up using a fixed volume pipet. Thesample then mixed with 990 ul of sample buffer. The blood and samplebuffer are mixed. Using a fixed volume pipet bulb, 100 ul is drawn upand pipetted onto the LFA cassette containing integrated intact C3 andiC3b lateral flow immunoassay cassettes. Alternatively, 100 ul can beapplied to separate intact C3 and iC3b cassettes. The cassette orcassettes will be placed in reader at the patient's bedside. The readeris set to take a reading after 20 minutes. Data is collected and iC3b,intact C3 and iC3b:(intact C3) values recorded at each time point.

Changes in intact C3 or iC3b levels over time or changes in the rate ofchange may indicate a change in inflammatory status. A sharp rise iniC3b, accompanied by a decrease in intact C3, indicates imminentrespiratory distress. As a next course of action, a clinician performs abroncheoalveolar lavage (BAL) on the patient to determine whether thepatient is experiencing VAP. If bacteria are present at levels of 104per ml or higher, VAP is indicated and the patient is placed onantibiotic therapy. Otherwise, noninfectious inflammatory dysfunction isassumed and the patient may be treated with anti-inflammatory agentsand/or complement inhibitors. The patient may also have his ventilatorsetting adjusted.

Example 3 Determining Disease Severity and Effectiveness of Treatment ina Patient with Systemic Lupus Erythematosus (SLE)

Before the first test sample is assayed, a standard curve is performedusing 10 ng/ml 30 ng/ml, 100 ng/ml, 300 ng/ml, and 1000 ng/ml of intactC3 and iC3b standards. Lateral flow immunoassay cassettes are read withan electronic reader after 20 minutes.

The test is used to gauge the initial severity of disease as well as theeffectiveness of therapy. One of the standard diagnostics performed onSLE patients is measurement of total C3 levels. C3 levels are normallydepressed in SLE patients and return to normal (>1 mg/ml) followingsuccessful treatment. However, it is not known generally whether the C3activation has been abrogated or only slowed enough to allow normalreplenishment mechanisms to restore C3 levels to normal.

At each doctor visit, a patient's blood is collected for total C3,intact C3, and iC3b tests. Only one drop is required for the combined 3tests. Blood is collected by fingerstick unless blood is being drawn forother tests, in which case, the blood will come from that source. A 10ul sample is drawn up using a fixed volume pipet. The sample then mixedwith 990 ul of sample buffer. The blood and sample buffer are mixed.Using a fixed volume pipet bulb, 100 ul is drawn up and pipetted ontothe LFA cassette containing integrated lateral flow cassette thatmeasures total C3, intact C3 and iC3b. Alternatively, 100 ul can beapplied to separate cassettes for each assay. The cassette or cassettesare placed in reader at the doctor's office. The reader is set to take areading after 20 minutes.

Data is collected at each doctor visit. At the initial visit, adding theiC3b and intact C3 tests provides the specialist with more informationabout the severity of the patient's condition than is now possible. Newinformation becomes available at the time that the specialist wouldconsider the patient's status stable. At this point, the iC3b and intactlevels indicate the extent of remaining disease process. If iC3b levels,in particular, are above normal (generally >1%), the underlying diseaseprocess is still very active and the specialist may opt to furtheradjust therapy by increasing anti-inflammatory drug doses or addingadditional medication.

Example 4 Determination of Basal Intact C3 and iC3b Levels in the BasalTear Fluid of a Healthy Individual Over a 24 Hour Period

Before the first test sample is assayed, a standard curve is performedusing 10 ng/ml, 30 ng/ml, 100 ng/ml, 300 ng/ml, 1000 ng/ml, and 3000ng/ml of intact C3 and iC3b standards. Lateral flow immunoassaycassettes are read with an electronic reader after 20 minutes.

For determining intact C3 and iC3b levels in the eye of a healthyindividual, three readings in total were taken. Samples were collectedand evaluated of Time=0 hours, 12 hours, and 24 hours.

For tear collection, the lower eyelid is pulled back and briefly dappedwith a Kimwipe® to the lower part of the eye. The Kimwipe® is thenquickly cut where the tear was collected leaving a few millimeters ofdry edge surrounding the tear spot. The Kimwipe®-tear sample is thenplaced into 220 ul of BioAssay Works Diluent Buffer and vortexedthoroughly for 10 seconds. After a one minute wait period, the sample isvortexed again briefly. Next, 100 ul of sample is transferred to eachlateral flow immunoassay (intact C3 and iC3b) and assayed.

For analysis, each cassette is inserted into the reader and read after20 minutes.

The results ranged between 50-60 μl/ml of intact C3 and between 5-8μl/ml of iC3b (See FIG. 15).

Example 5 Determination of Basal Intact C3 and iC3b Levels in Two HealthIndividuals at a Single Time Point

Before the first test sample is assayed, a standard curve is performedusing 10 ng/ml, 30 ng/ml, 100 ng/ml, 300 ng/ml, 1000 ng/ml, and 3000ng/ml of intact C3 and iC3b standards. Lateral flow immunoassaycassettes are read with an electronic reader after 20 minutes.

Resting levels of intact C3 and iC3b are collected from two healthydonors. The lateral flow immunoassay reader is turned on. Finger iscleaned using an alcohol swab. Finger is stuck with lancet and squeezedgently to collect 10 ul of blood using the MICROSAFE® Tube by capillaryaction. Blood sample was expelled directly into a tube filled with 990ml of sample assay buffer and then capped and mixed by inversion 6-8times. 100 ul of blood sample mixture was transferred to CompAct intactC3 test using the 100 ul Exact Volume Pipet. A second 100 ul of bloodsample mixture was then transferred to the CompAct iC3b test using afresh 100 ul Exact Volume Pipet. The timer was set to read after 20minutes for both tests.

The results from the first patient were determined to be approximately500 μg/ml for intact C3 and 200 ng/ml for iC3b. This indicates there is2500 ratio of intact C3 to iC3b in this individual (see FIG. 16). Thesecond individual's results were approximately 1000 μg/ml for intact C3and 300 ng/ml for iC3b (see FIG. 17). Both of these values are withinthe expected normal ranges. The iC3b values are in the lower range ofwhat is considered normal.

Example 6 Determination of Basal Intact C3 and iC3b Levels in a HealthyIndividual at a Single Time Point after Strenuous Exercise

Using the above protocol of Example 5, one of the healthy individualswas tested again after strenuous exercise (see FIG. 18). Exertion didnot significantly alter iC3b or C3 levels.

Example 7 Crosstalk Between Intact C3 and iC3b Antibodies in LateralFlow Immunoassays

In a 1 milliliter volume, 50 ng/ml of iC3b was mixed with varyingamounts of intact C3 (ranging from 0 ng/ml to 100,000 ng/ml). Sampleswere mixed by inversion 6-8 times and then 0.1 ml was pipetted onto thecassette. Readings were taken at 20 minutes. Reader output was convertedto iC3b concentration using a standard curve generated from 10 ng/ml to100,000 ng/ml. Background from a cassette run only with buffer wassubtracted. Fractional contributions were calculated by subtracting theactual iC3b concentration (from iC3b test with no added C3) fromapparent concentration of iC3b at each point and then normalizingagainst actual iC3b concentrations. See FIG. 14. For the H08K-01cassettes, at the highest concentration of C3 tested, about half theiC3b signal came from intact C3 and half from actual iC3b. For theJ24K03 version, about four times as much iC3b signal came from intact C3cross talk than from actual iC3b. Although these antibody pairs workwell in ELISA assays, they exhibit significant crosstalk when used inlateral flow immunoassays for the same analytes. At the physiologicallyrelevant 250:1 and 500:1 ratios, intact C3 contributes more to iC3bsignal output than iC3b itself in J24K-03.

J24K-03 is an assay with mouse anti-C3a monoclonal on the gold conjugateand mouse anti-C3d monoclonal on the test line. H08K-01 has mouseanti-iC3b monoclonal on the gold conjugate and Anti-C3 polyclonal on thetest line.

Example 8 Generation of iC3b Standard Curve for Lateral FlowImmunoassays

One embodiment of the invention comprises a lateral flow assay stripwithout the cassette casings. These strips had anti-iC3b monoclonal(Quidel® A209) conjugated to the gold and anti-C3 antibody (MPBiomedical® 55237) conjugated to the strip. Standard curves are shown inFIG. 11(A). The standard curves indicated a linear range of about 10fold and a sensitivity of about 100 ng/ml. Another embodiment of theinvention configures the strips for use in a cassette that allowscontrolled application of the sample to the assay strip. This improvedassay-to-assay reproducibility, although there is still considerabletime dependence on the assay. Standard curve results are shown in FIG.11(B). A third embodiment increases the antibody concentration from 0.5mg/ml to 1 mg/ml applied to on the gold conjugate and removes BSA fromthe absorption buffer. Standard curve results are shown in FIG. 11(C).

Standard curves are generated as described in example 9 below.

Example 9 Generation of iC3b Standard Curve for Lateral FlowImmunoassays

Ten (10) μl of a stock of iC3b (concentration 1 mg/ml) was diluted into990 ul of sample dilution buffer to create 10 ug/ml working stock usinga 2 ml capped tube. Tube was mixed by slowly inverting 10-12 times.Investigator diluted 500 ul of 10 ug/ml stock into 500 ul BAW Buffer tocreate a 5 ug/ml stock in another 2 ml capped tube. Mixing was performedby slowly inverting tube 10-12 times. The 1:1 dilution (500 ul:500 ul)was repeated, as described above, nine more times to create thefollowing working stocks: 10 ug/ml, 5 ug/ml, 2.5 ug/ml, 1.25 ug/ml, 625ng/ml, 313 ng/ml, 156 ng/ml, 78 ng/ml, 39 ng/ml, 20 ng/ml, 10 ng/ml, and0 ng/ml (buffer alone).

Lateral flow immunoassay (LFA) cassettes were prepared by labeling andlaying out in groups of three. For each dilution, investigator pipetted100 ul of first working stock (10 ug/ml for intact C3 and 5 ug/ml foriC3b) into sample port of 1st LFA. For each concentration, investigatorwaited 20 seconds before loading 100 ul of same working stock into the2nd LFA. Cassettes were read after 10, 20, and 30 minutes using BioAssayWorks Reader LFDR 101 (Forsite Diagnostics) using the Test line settingfollowed by the Control line setting, and the data recorded.

After the experiment is completed, data was plotted using GraphPad Prism5 software. The standard curve fits the three-parameter logisticequation: Y=Bottom+(Top−Bottom)/(1+EC50/X). See FIG. 12.

Example 10 Generation of Intact C3 Standard Curve for Lateral BowImmunoassays

Ten (10) μl of a stock of intact (concentration 1 mg/ml) was dilutedinto 990 ul of sample dilution buffer to create 10 ug/ml working stockusing a 2 ml capped tube. Tube was mixed by slowly inverting 10-12times. Investigator diluted 500 ul of 10 ug/ml stock into 500 ul BAWBuffer to create a 5 ug/ml stock in another 2 ml capped tube. Mixing wasperformed by slowly inverting tube 10-12 times. The 1:1 dilution (500ul:500 ul) was repeated, as described above, nine more times to createthe following working stocks:

10 ug/ml, 5 ug/ml, 2.5 ug/ml, 1.25 ug/ml, 625 ng/ml, 313 ng/ml, 156ng/ml, 78 ng/ml, 39 ng/ml, 20 ng/ml, 10 ng/ml, and 0 ng/ml (bufferalone).

LFA cassettes were prepared by labeling and laying out in groups ofthree. For each dilution, 100 ul of first working stock (10 ug/ml forintact C3 and 5 ug/ml for iC3b) was pipetted into sample port of 1stLFA. For each concentration, investigator waited 20 seconds beforeloading 100 ul of same working stock into the 2nd LFA. Cassettes wereread after 10, 20, and 30 minutes using BioAssay Works Reader LFDR 101(Forsite Diagnostics) using the Test line setting followed by theControl line setting, and the data recorded.

After the experiment is completed, data was plotted using GraphPad Prism5 software. The standard curve fits the three-parameter logisticequation: Y=Bottom+(Top−Bottom)/(1+EC50/X). See FIG. 13.

Example 11 Embodiment of a CompAct™ Lateral Flow Assay Procedure

FIG. 21 depicts an exemplary embodiment of a lateral flow formatimmunoassay. Initially, a finger of a patient is cleaned with an alcoholswab and the finger is pricked with a lancet. The finger is thensqueezed to facilitate presentation of blood, which is then collected ina Microsafe™ tube. The blood is expelled from the Microsafe™ tubedirectly into a sample port of a CompAct™ test cassette. Next, threedrops of assay buffer are added into the sample port to mix with anddilute the blood sample. The samples are allowed to sit for a period oftime, for example 15 to 20 minutes. If desired, a second blood samplemay be drawn as described and the sample then introduced to a secondCompAct™ test cassette (“B”). At some point at or before the end of thedesired time period, each test cassette may be visually inspected to besure that the control line is readily visible and that smearing of thesample is not likely to cause a problem in reading the data. Once thedesired time period has elapsed, each test cassette may be introduced toa reading device to capture data from each test, for example, as shownin FIG. 21. Also shown in FIG. 21 are conceptual sketches of thecleaning and sample extraction procedures as well as photographs of testcassettes and a test reader contemplated as within the scope of thepresent invention.

Example 12 Sensitivity of CompAct™ Lateral Flow Assay Embodiment forHuman C3

Ten (10) μl of a stock of purified human C3 protein (ComplementTechnology, Inc., #A113, concentration 1 mg/ml) was diluted into 990 ulof Sample Diluent Buffer (BioAssay Works, ISOT-003) to create 10 ug/mlworking stock in a 2 ml capped tube. The working stock was mixed byslowly inverting 10-12 times. Investigator diluted 500 ul of 10 ug/mlstock into 500 ul BAW Buffer to create a 5 ug/ml stock in another 2 mlcapped tube. Mixing was performed by slowly inverting tube 10-12 times.The 1:1 dilution (500 ul:500 ul) was repeated, as described above, ninemore times to create the following working stocks: 10 ug/ml, 5 ug/ml,2.5 ug/ml, 1.25 ug/ml, 625 ng/ml, 313 ng/ml, 156 ng/ml, 78 ng/ml, 39ng/ml, 20 ng/ml, 10 ng/ml, and 0 ng/ml (buffer alone).

Lateral flow assay (LFA) cassettes were prepared by labeling and layingout in groups of three. Each set of triplicate tests were photographed(Nikon D80 with 60 mm AF Micro Nikkor lens), and the images wereassembled into a single image using a Paint application. The photographsof each replicate can be seen in FIG. 22.

For each dilution, 100 ul of first working stock (10 ug/ml for intact C3and 5 ug/ml for iC3b) was pipetted into sample port of a first LFA. Foreach concentration, investigator waited 20 seconds before loading 100 ulof same working stock into the 2nd LFA. Cassettes were read after 10,20, and 30 minutes using BioAssay Works Reader LFDR-001 (ForsiteDiagnostics) using the Test line setting followed by the Control linesetting, and the data recorded. The Sandwich value for each time pointwas calculated as a percentage: [Test line]/[Control line]×100% and theresults are shown in FIG. 23. As assessment of variability of the assaysused in this example was calculated and is shown in FIG. 24.

Example 13 Sensitivity of CompAct™ Lateral Flow Assay Embodiment forHuman iC3b

Ten (10) μl of a stock of purified human iC3b protein (ComplementTechnology, Inc., #A115, concentration 1 mg/ml) was diluted into 990 ulof sample diluent buffer (BioAssay Works, ISOT-003) to create a 10 ug/mlworking stock in a 2 ml capped tube. The working stock was mixed byslowly inverting 10-12 times. Investigator diluted 500 ul of 10 ug/mlstock into 500 ul BAW Buffer to create a 5 ug/ml stock in another 2 mlcapped tube. Mixing was performed by slowly inverting tube 10-12 times.The 1:1 dilution (500 ul:500 ul) was repeated, as described above, ninemore times to create the following working stocks: 10 ug/ml, 5 ug/ml,2.5 ug/ml, 1.25 ug/ml, 625 ng/ml, 313 ng/ml, 156 ng/ml, 78 ng/ml, 39ng/ml, 20 ng/ml, 10 ng/ml, and 0 ng/ml (buffer alone).

Lateral flow immunoassay (LFA) cassettes were prepared by labeling andlaying out in groups of three. Each set of triplicate tests werephotographed (Nikon D80 with 60 mm AF Micro Nikkor lens), and the imageswere assembled into a single image using a Paint application. Thephotographs of each replicate can be seen in FIG. 25.

For each dilution, investigator pipetted 100 ul of first working stock(10 ug/ml for intact C3 and 5 ug/ml for iC3b) into sample port of 1stLFA. For each concentration, investigator waited 20 seconds beforeloading 100 ul of same working stock into the 2nd LFA. Cassettes wereread after 10, 20, and 30 minutes using BioAssay Works Reader LFDR-001(Forsite Diagnostics) using the Test line setting followed by theControl line setting, and the data recorded. The Sandwich value for eachtime point was calculated as a percentage: [Test line]/[Controlline]×100% and the results are shown in FIG. 26.

Example 14 Assessment of Human Native C3 and iC3b Using a Lateral FlowAssay Embodiment

Whole blood was collected via a finger stick using a safety lancet(Fisher Healthcare, 02-675-160) and transferred via a pipet to a 2 mltube. Next, 30 ul of blood was immediately diluted into 270 ul SampleDiluent Buffer (BioAssay Works, ISOT-003) in a separate 2 ml tube, and100 ul of the 1:10 blood dilution was applied to the iC3b LFA. The 1:10blood dilution was further diluted into Sample Diluent Buffer to a finalconcentration of 1:3162, and 100 ul was applied to the C3 LFA. The Testand Control lines were read for each test at 20 and 30 minutes using areader (Forsite, #LFDR-001) and triplicate samples were run for eachdata point. The estimated concentration of native C3 and iC3b werederived using the Sandwich value for the sample and the standard curvefor the particular assay. The data for these assays is shown in FIG. 27.

Example 15 Assessment of Human C3 Levels from Samples of Human WholeBlood, Plasma, and Serum

This study analyzed human donor samples of whole blood, plasma, andserum for levels of native C3 and levels of iC3b. Three donors hadsamples taken at three different time points during a one week period atdays 1, 2, and 5. The procedures used to extract the samples andintroduce them to the cassettes were substantially as described inExample 11. Whole blood samples were tested immediately after extractionvia a CompAct™ lateral flow assay test, while plasma and serum sampleswere prepared and tested either via a CompAct™ lateral flow assay testor a standard ELISA assay.

For samples tested with a CompAct™ lateral flow assay, plasma-EDTA andserum were prepared from fresh whole blood per manufacturer's protocols(Becton, Dickinson and Company) and frozen in aliquots at −80° C.Aliquots were thawed on ice and assayed via the CompAct™ assay.Estimated iC3b concentrations were derived from purified proteinstandard curves.

For samples tested in a standard ELISA assay, the following procedureswere used. First, 96 well plates were coated using 50 ul of monoclonalantibody diluted to 2 ug/ml in 1×PBS. For the human C3 ELISA assays,Immnulon 4HBX plates (Thermo Scientific, 3855) were coated with mouseanti-human C3/C3a monoclonal antibody (Cell Sciences, HM2075). For thehuman iC3b ELISA assays, Immulon 1B 96-well plates (Thermo Scientific,3355) were coated with mouse anti-human iC3b monoclonal antibody(Quidel, A209). The ELISA plates were allowed to incubate at roomtemperature for 1-2 hours. The liquid containing the monoclonal antibodywas discarded and each well was washed twice with 1×PBS-Tween 0.05%(PBS-T). Next, the wells were blocked with 200 ul of StartingBlockbuffer (Thermo Scientific, 37538) and incubated for one hour at roomtemperature. The buffer was then discarded and cells were washed threetimes with 1×PBS-T. The purified protein standards were diluted into1×PBS to a concentration of 1 ug/ml in a separate microtiter plate(Thermo Fisher #9205). The human C3 standard used was from ComplementTechnology, Inc (#A113) and the human iC3b standard used was also fromComplement Technology, Inc. (#A115). Each standard was then seriallydiluted using equal volumes of 1×PBS.

Each plasma and serum sample was prepared from whole blood as describedabove, and then diluted 1:20 in 1×PBS into the separate microtiter plateand then serially diluted using 1×PBS. Then, 50 ul of each dilutedsample was transferred into the appropriate well of the ELISA plate andallowed to incubate at room temperature for one hour. The liquid wasthen discarded and each well was washed six times using 1×PBS-T. Fifty(50) ul of goat anti-C3-HRP (MP Biomedicals, #55237) was diluted 1:2000into StartingBlock buffer and added to each well of the ELISA plate. Thesamples were then incubated for one hour at room temperature. The liquidwas then discarded and each well was washed six times using 1×PBS-T.Fifty (50) ul of a 1:1 mixture of TMB horse radish peroxidase substrate(Thermo Fisher #1854060 and #1854050) were added to each well. Afterthree minutes, 25 ul of 1M H₂SO₄ was added to each well to stop thereaction. The ELISA plate was then analyzed using a BMG LabtechPOLARstart Omega plate reader at 450 nm. The results of theseexperiments can be seen in FIGS. 28 and 29.

FIG. 28 shows that levels of human C3 protein assessed using a CompAct™lateral flow assay embodiment of the invention generally match thoselevels observed in the literature that were generated using ELISAmethods.

FIG. 29 shows that the levels of iC3b protein detected in plasma andserum samples varies significantly between the CompAct™ lateral flowassay embodiment and previously known ELISA methods. The data shown in(A), (B), and (C) are shown as bar graphs by donor, while the data in(D) is shown numerically in a table. This was a surprising result thathighlights the advantages of the methods and assays of the presentinvention. Without wishing to be held to a particular theory, applicantspropose that the significantly lower levels of iC3b assayed using theCompAct assay may be due to the rapid speeds at which data is generatedversus the much slower and more ELISA methods. It is possible thatshortening reaction times does not allow for factors in a sample to acton and potentially activate complement proteins. The increased number ofmanipulating and handling steps required by the ELISA may also becontributing to these results in a similar manner, buy facilitatinginteraction between intact C3 and activating agents, for example.

This hypothesis is supported as shown in FIG. 30 where it is clear thatwhen purified samples of iC3b are used, both the CompAct™ and ELISAtests produce similar data. However, when samples derived from bodyfluid are used, the result vary significantly. Regardless of themechanism behind the results, such a large difference in detected levelsof iC3b in the same sample may easily lead to erroneous clinicaldiagnoses, highlighting one of the advantages of embodiments of thepresent invention.

Example 16 The Effect of Time on Detected iC3b Levels

In an effort to assess the effects of time on the levels of iC3bdetected in samples exposed to room temperature using the CompAct™assay, diluted samples of whole blood, plasma, and serum obtained inExample 15 were re-tested after four hours of time at room temperature.Other assay methods and conditions were the same as described in Example15 and the data is shown in FIG. 31. The data clearly shows that thereis an approximately 100-fold increase in the levels of iC3b in eachsample due to the passage of time at room temperature. This previouslyunknown effect strongly indicates that substantial increases in iC3blevels occur rapidly at room temperature. This further highlights anadvantage of embodiments of the present invention and the potentialfalse data generated by previous methods, including ELISA assays, whichrequire hours of time to generate data.

In an effort to achieve better resolution in terms of the increases ofC3 activation over time in various samples, aliquots of whole blood,plasma, and serum samples obtain in Example 15 above were assessed atdifferent time points to determine how quickly C3 activation may beobserved.

FIG. 32 shows levels of iC3b detected using a CompAct™ assay embodimentof the invention in samples of whole blood as compared to samplescontaining purified iC3b protein standards. Samples were tested after 5,10, 15, or 20 minutes of exposure to room temperature and the assayswere run substantially as described in example 15. (A) shows that someincreases in iC3b may be detected in whole blood after only 5 minutes atroom temperature, and the magnitude of this increase appears toaccelerate over time as can be seen by the increasing slope of the datain (B), (C) and (D).

FIG. 33 shows the difference in iC3b levels detected in a CompAct™ assayembodiment of the invention after either 5 minutes or 60 minutesexposure to room temperature. The samples of plasma and serum wereobtained and prepared as described above for example 15. As can be seenin FIG. 33, the increase in iC3b levels detected was far more robust at60 minutes than 5 minutes. Additionally and unexpectedly, the increasesin iC3b levels does not appear to uniformly increase over time, asevidenced by the significant alterations in slope over time. Thisphenomenon adds an additional layer of complexity to the proper analysisof complement activation and further highlights the importance ofminimizing assay time and processing. Several embodiments of the presentinvention address this previously unknown problem in the detection ofcomplement activation.

Example 17 Time Dependent Generation of iC3b in Human Plasma

In this example, human plasma from a healthy donor was diluted 1:10 intoSample Diluent Buffer and immediately assay via iC3b LFA strips. Thediluted plasma continued to incubate at room temperature and was testedafter an additional incubation time of 30 min, 1 hr, 2 hr, 4 hr and 72hr (3 days). The Test and Control lines for each time point were read bya reader (Forsite, #LFDR-001) 30 minutes after application of the sampleto the LFA. The Sandwich value was calculated as a percentage (Testline/Control line×100%) after subtracting out Test line signal from abuffer-alone sample (not shown). FIG. 34 shows the time dependentactivation of iC3b in plasma, with (A) showing the test strips afterincubation and (B) showing the data as read by the Forsite reader. Thegraph of (B) is plotted with hours on the X-axis and Sandwich value onthe Y-axis.

Example 18 Time Dependent Generation of iC3b in Human Plasma and SerumUsing ELISA

In this example, as shown in FIG. 35, human plasma and serum from ahealthy donor was serially diluted into ELISA Buffer and incubated withwells coated with anti-iC3b monoclonal antibody for either (A) 2 minutesor (B) 5 minutes before stopping the incubation by removal and washing.Then, all the wells were incubated with HRP conjugated anti-C3polyclonal antibody for detection. Purified iC3b protein was alsodiluted and incubated side-by-side with the plasma and serum samples forthe times indicated to generate a standard curve for each time point asis shown in FIG. 35. The concentration of iC3b in the diluted serum wasderived from the corresponding iC3b standard curve for each time point.

As FIGS. 35 (A) and (B) show, activation of iC3b in human plasma andserum can occur very quickly, even as quickly as two minutes in somecases. The activation of iC3b in human serum was particularly strong inthis example and highlights the dramatic effect time can have on samplesanalyzed using ELISA type assays.

In another experiment, human serum from a healthy donor was seriallydiluted into ELISA Buffer and incubated with wells coated with anti-iC3bmonoclonal antibody for the times indicated before stopping theincubation by removal and washing. Then, all the wells were incubatedwith HRP conjugated anti-C3 polyclonal antibody for detection. PurifiediC3b protein was also diluted and incubated side-by-side with thediluted serum for the times indicated to generate a standard curve foreach time point. The concentration of iC3b in the diluted serum wasderived from the corresponding iC3b standard curve for each time point.FIG. 36 shows the relative levels of iC3b detected over time in thesesamples, with a doubling of iC3b levels shown by as little as 15 minutesof incubation.

Example 19 C3 Activation in the Absence of Antigen:Antibody Complexes

After coating wells with anti-ovalbumin antibody (does not recognize C3or iC3b) and blocking with StartingBlock Buffer (Pierce 37538), humanserum was diluted into Veronal Buffer (Lonza, 12-624E) in the wells andallowed to incubate for either 10 or 60 minutes+/−10 mM EDTA. Afterwashing, the wells were incubated for 60 minutes with HRP-conjugatedanti-C3 polyclonal antibody for detection using Peroxide Solution(Thermo Sci., 1854060) and Peroxidase Substrate TMB (Thermo Sci.,1854050). The wells were read for absorbance at 450 nm.

FIG. 37 shows that the serum-derived C3 in this experiment wassignificantly activated after 10 minutes or 60 minutes of incubation onthe OVA coated plate. Without wishing to be held to a specific theory,it appears that this activation is via an EDTA-sensitive activationpathway. It is noteworthy that the deposition of C3 occurred ontoanti-ovalbumin antibody coated wells in the absence of ovalbumin,showing that an ovalbumin: ovalbumin antibody complex is not requiredfor C3 activation and deposition. This highly unexpected result meansthe observed C3 deposition is likely not occurring via a traditionalcapture phenomenon that is normally associated with an ELISA assay.

Example 20 C3 Activation in the Absence of Antibody-Coated Wells

Wells were blocked with StartingBlock Buffer (in the absence ofantibody), human serum was diluted into Veronal Buffer (Lonza, 12-624E)in the wells and allowed to incubate for either 10 or 60 minutes+/−10 mMEDTA. After washing, the wells were incubated for 60 minutes withHRP-conjugated anti-C3 polyclonal antibody for detection using PeroxideSolution (Thermo Sci., 1854060) and Peroxidase Substrate TMB (ThermoSci., 1854050). The wells were read for absorbance at 450 nm.

FIG. 38 shows that human serum-derived C3 is apparently activated via anEDTA-sensitive pathway and deposited onto wells blocked with buffer inthe absence of antibody. Thus, not only is an antigen:antibody complexnot required for C3 activation and deposition, as shown in Example 19,but not even an antibody is required to observe this phenomena. Much aswith Example 19 above, this highly unexpected result indicates that theC3 deposition observed is not a capture phenomenon that is normallyassociated with ELISA immunodetection.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to one skilled in the artthat various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is, therefore,intended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed is:
 1. A method comprising steps of detecting in asample from a subject a first level of iC3b protein wherein thedetecting involves specific interaction between the iC3b protein and adetecting agent; comparing the detected level with a reference level,which reference level is within a range of about 10 ng/ml to about 5,000ng/ml; wherein determination that the detected level is above thereference level indicates that the subject is suffering from orsusceptible to undesirable and/or pathologic complement activation; andadministering a treatment or implementing a change in treatment to treatundesired and/or pathologic complement activation if the detected levelis above the reference level.
 2. The method of claim 1, furthercomprising detecting in a sample from a subject a second level of iC3bprotein wherein the detecting involves specific interaction between theiC3b protein and a detecting agent; and administering a treatment orimplementing a change in treatment to treat undesired and/or pathologiccomplement activation if the second level of iC3b protein is within orbelow the reference level.
 3. The method of claim 1, wherein thedetecting is completed within about 2 hours or less from the time thesample is taken from the subject.
 4. The method of claim 1, wherein thedetecting is completed within about 60 minutes or less from the time thesample is taken from the subject.
 5. The method of claim 1, wherein thedetecting is completed within about 30 minutes or less from the time thesample is taken from the subject.
 6. The method of claim 1, wherein thedetecting agent is a non-cross reactive antibody to iC3b.
 7. The methodof claim 6, wherein the non-cross-reactive antibody is sufficientlynon-cross-reactive with intact C3 that a solution concentration of atleast 1 ug intact C3/ml would be required to generate a level of bindingsignal comparable to that observed for the antibody with a 1 ng/mlsolution of iC3b.
 8. The method of claim 6, wherein thenon-cross-reactive antibody binds to a neoepitope of iC3b protein. 9.The method of claim 1, wherein the reference level is within a range ofabout 100 ng/ml to 3,000 ng/ml.
 10. The method of claim 1, wherein thereference level is within a range of about 200 ng/ml to 1,000 ng/ml. 11.The method of claim 1, wherein the undesirable and/or pathologiccomplement activation is caused by a disorder selected from the groupconsisting of trauma, inflammatory stress, autoimmune disorders,intracranial hemorrhage, infection, transplant rejection, oculardisease, heart disease, ischemia/reperfusion injury, paroxysmalnocturnal hemoglobinuria, hereditary angiodema, renal disease,pregnancy-associated disorders, and neurological disorders.
 12. Themethod of claim 1, wherein the sample is selected from the groupconsisting of whole blood, serum, plasma, urine, tears, saliva, woundexudate, bronchoalveolar lavage fluid, and cerebrospinal fluid.
 13. Themethod of claim 1, wherein the step of detecting is performed undercontrolled conditions such that performance of the step does notsubstantially activate complement within the sample.
 14. The method ofclaim 1, wherein an ELISA assay is not utilized in either of thedetecting or comparing steps.
 15. The method of claim 1, wherein thedetecting and comparing steps are carried out using a lateral flowassay.
 16. The method of claim 1, wherein the detecting and comparingsteps are repeated at least once at least two hours after theperformance of the first detecting and comparing steps.
 17. A methodcomprising steps of detecting in a sample from a subject a first levelof iC3b protein wherein the detecting involves specific interactionbetween the iC3b protein and a detecting agent; detecting in a samplefrom a subject a second level of iC3b protein wherein the detectinginvolves specific interaction between the iC3b protein and a detectingagent; comparing the second level of iC3b protein to the first level ofiC3b protein; wherein determination that the second level of iC3bprotein is at least 25% above the first level of iC3b protein indicatesthat the subject is suffering from or susceptible to undesirable and/orpathologic complement activation; and administering a treatment orimplementing a change in treatment to treat undesired and/or pathologiccomplement activation if the second level of iC3b protein is at least25% above the first level of iC3b protein.
 18. The method of claim 17,further comprising detecting in a sample from a subject a third level ofiC3b protein wherein the detecting involves specific interaction betweenthe iC3b protein and a detecting agent; and administering a treatment orimplementing a change in treatment to treat undesired and/or pathologiccomplement activation if the third level of iC3b protein is at least 25%below the second level of iC3b protein.
 19. The method of claim 17,wherein at least one detecting step is completed within about 2 hours orless from the time the sample is taken from the subject.
 20. The methodof claim 17, wherein at least one detecting step is completed withinabout 60 minutes or less from the time the sample is taken from thesubject.
 21. The method of claim 17, wherein at least one detecting stepis completed within about 30 minutes or less from the time the sample istaken from the subject.
 22. The method of claim 17, wherein thedetecting agent is a non-cross reactive antibody to iC3b.
 23. The methodof claim 22, wherein the non-cross-reactive antibody is sufficientlynon-cross-reactive with intact C3 that a solution concentration of atleast 1 ug intact C3/ml would be required to generate a level of bindingsignal comparable to that observed for the antibody with a 1 ng/mlsolution of iC3b.
 24. The method of claim 22, wherein thenon-cross-reactive antibody binds to a neoepitope of iC3b protein. 25.The method of claim 17, wherein the undesirable and/or pathologiccomplement activation is caused by a disorder selected from the groupconsisting of trauma, inflammatory stress, autoimmune disorders,intracranial hemorrhage, infection, transplant rejection, oculardisease, heart disease, ischemia/reperfusion injury, paroxysmalnocturnal hemoglobinuria, hereditary angiodema, renal disease,pregnancy-associated disorders, and neurological disorders.
 26. Themethod of claim 17, wherein the sample is selected from the groupconsisting of whole blood, serum, plasma, urine, tears, saliva, woundexudate, bronchoalveolar lavage fluid, and cerebrospinal fluid.
 27. Themethod of claim 17, wherein the step of detecting is performed undercontrolled conditions such that performance of the step does notsubstantially activate complement within the sample.
 28. The method ofclaim 17, wherein an ELISA assay is not utilized in either of thedetecting or comparing steps.
 29. The method of claim 17, wherein thedetecting and comparing steps are carried out using a lateral flowassay.
 30. The method of claim 17, wherein the detecting and comparingsteps are repeated at least once at least two hours after theperformance of the first detecting and comparing steps.